Download Abstract Book 2012 10th Annual Meeting May 23 - 25, 2012

Abstract Book 2012
10th Annual Meeting
May 23 - 25, 2012
Rheingoldhalle Congress Center
Mainz, Germany
Abstract Book sponsored by:
CIMT | Summary
Summary
Pages:
Chapter title:
005
006-007
008
Introduction
Who We Are
Websites
010
011-018
2012 Program Committee
2012 Speakers
019-021
022-025
026-029
Program Schedule CIMT 2012 | Day 1 | May 23
Program Schedule CIMT 2012 | Day 2 | May 24
Program Schedule CIMT 2012 | Day 3 | May 25
030
031
Floor Plan
Program Overview
032
033
Sponsors, Partners & Supporters
Industry Exhibitors
034
Poster Presentations
036-047
Abstract List
050-093
094-137
138-164
165-197
198-234
235-271
Cellular Therapy
Improving Immunity
Immunomonitoring
New Targets & New Leads
Therapeutic Vaccination
Tumor Biology & Interaction with the Immune System
272
Imprint
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CIMT | Introduction
Dear colleagues, members and friends,
Towards Next-Generation
Cancer Immunotherapy
It is with great satisfaction and joy that we are welcoming you to the 10th annual meeting of
the Association of Cancer Immunotherapy. When CIMT was founded 10 years ago, a small group
of immunologists and oncologists saw the need for a European forum to exchange knowledge and
expertise on the emerging discipline of cancer immunotherapy. There was a lot to learn, a lot to
explore and the desire to connect with pioneering researchers worldwide. Since then, the CIMT
annual meetings have steadily grown in scope and attendance, keeping pace with the field of
immuno-oncology.
We are proud to have given many young researchers the chance to present their work for the first
time. We are honored that so many acknowledged experts have come to CIMT and shared their
wisdom. We thank you all for your dedication and passion to develop new and better therapies for
people suffering from cancer.
CIMT 2012 will provide a stage for reflecting the aggregate knowledge of 10 years of cancer
immunotherapy, presenting current scientific approaches and discussing next-generation therapies.
Highlights of the 10th anniversary meeting will be individualized therapy approaches and therapeutic antibodies. Sessions in therapeutic vaccination, cellular therapy and improving immunity will
discuss recent advances and future applications. Immunomics, combining immunology with genomics, transcriptomics, systems biology, and bioinformatics, is having an increasing impact on the
development and testing of immunotherapies. A dedicated session will highlight new methods, tools,
and results that are advancing our ability to generate immunotherapies and treat cancer patients.
In addition, CIP (CIMT Immunoguiding Program) and RRG (Regulatory Research Group) will hold
sessions and panel discussions focusing on standardized and innovative tools for improving the
quality of immunomonitoring and facilitating the translation of scientific knowledge into drug
development.
We look forward to an exciting anniversary meeting and the next decade of innovations in cancer care.
The CIMT Executive Board
Ch. Huber (Mainz)
H.G. Rammensee (Tübingen)
P. Johnson (Southampton)
C. Figdor (Nijmegen)
U. Kalinke (Hanover)
D. Schendel (Munich)
C. Melief (Leiden)
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CIMT | Who We Are
CIMT | Who We Are
Who We Are
Who We Are
The Association
Executive Board:
The Association for Cancer Immunotherapy (CIMT) was founded in
2002 as an information and education platform for the emerging field
of immunological cancer therapy. Physicians and researchers from different fields of clinical and theoretical medicine were the founding members of the association. CIMT has since become the leading European
communication platform for all aspects of science and translation in the
field of cancer immunotherapy. CIMT operates as an independent nonprofit organization in Mainz, Germany, and is financed by donations,
sponsoring and congress fees.
Our Goals
Christoph Huber, chair
Sebastian Kreiter
Mainz, Germany
Mainz, Germany
Peter Johnson
Southampton, UK
offering a platform for knowledge exchange between scientists of
all faculties interested in cancer immunotherapy. CIMT connects industry-based scientists, academic scientists, regulatory authorities and physicians alike.
* cooperating internationally with partners in related consortia,
regulatory agencies, journals, academic institutions and companies.
* initiating working groups that actively accelerate the development in
the field.
Executive Director - Translational Medicine:
Ulrich Kalinke
Cedrik M. Britten
Hanover, Germany
Mainz, Germany
Cornelis Melief
Leiden, Netherlands
CIMT promotes the development of cancer immunotherapies by
*
Executive Director - Science:
Scientific Secretary
Hans-Georg Rammensee
Mustafa Diken
Tübingen, Germany
Mainz, Germany
Carl Figdor
Nijmegen, Netherlands
Executive Director - Communications:
Dolores Schendel
Christine Castle
Munich, Germany
Mainz, Germany
* organizing annual meetings, advanced education seminars,
symposia, workshops, and by publishing guidelines and textbooks.
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CIMT | Websites
Websites
visit our websites
www.cimt.eu & www.meeting.cimt.eu
find us on facebook
www.facebook.com/cimtmainz
follow us on twitter
www.twitter.com/C_IMT
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CIMT | Program Committee
CIMT | Speakers
2012 Program Committee
2012 Speakers
Axel Hoos
Dolores Schendel
Hinrich Abken
Tobias Raum
Co-Chairman, CIC Executive Committee;
Program Leader in Immunology/Oncology
at GlaxoSmithKline
GlaxoSmithKline
Philadelphia, USA
Institute for Molecular Immunology (IMI)
Helmholtz Zentrum
Munich, Germany
University of Cologne
Amgen Research
Cologne, Germany
Munich, Germany
Wolfgang Herr
Matthias Theobald
3rd Medical Department
University Medical Center of the
Johannes Gutenberg University
Mainz, Germany
3rd Medical Department
University Medical Center of the Johannes
Gutenberg University
Mainz, Germany
Sebastian Kreiter
Cedrik M. Britten
Translational Oncology (TRON) at the
University Medical Center of the
Johannes Gutenberg University
Mainz, Germany
Ribological GmbH
Mainz, Germany
Cécile Gouttefangeas
Mustafa Diken
University of Tübingen
Tübingen, Germany
Translational Oncology (TRON) at the
University Medical Center of the Johannes
Gutenberg University
Mainz, Germany
Ulrich Kalinke
Hansjörg Schild
Twincore
Hanover, Germany
Research Center Immunology (FZI) at the
Johannes Gutenberg University Mainz
Mainz, Germany
Hinrich Abken is Professor for Genetics & Immunology at CMMC
(Center for Molecular Medicine Cologne) at the University of
Cologne and Dept I Internal Medicine, Oncology-Hematology at
the University Hospital Cologne where he is working towards
the development of adoptive cell therapy of malignant diseases
using engineered T-cells. Dr. Abken's group made significant
contributions in the field of chimeric antigen receptors, recombinant targeting molecules to redirect T-cells towards defined
targets. The current projects are aimed at immunotherapy of
melanoma and gastrointestinal carcinomas, the development of
novel strategies in modulating an immune response and translation into clinical trials.
Shizuo Akira
WPI Immunology Frontier
Research Center
Osaka, Japan
Shizuo Akira, M.D., PhD, is Director of the WPI Immunology Frontier Research Center at Osaka University as well as
a member of the National Academy of Sciences and European
Molecular Biology Organization. Dr. Akira has received a number of prestigious awards including Robert Koch Prize, William
B. Coley Award, and Keio International Medical Science Prize.
Dr. Akira has made ground-breaking discoveries in the field
of immunology, most significantly in the area of innate host
defense mechanisms. Among his discoveries is the demonstration, through the ablation of toll-like receptor (TLR)s genes, that
TLRs recognize a discrete collection of molecules of microbal
origin, and later the RNA helicases, RIG-I (retinoic-acid-inducible protein I) and MDA5 (melanoma differentiation-associated
protein 5).
James P. Allison
Sloan-Kettering Institute
New York, USA
James P. Allison is Chair of the Immunology Program in the
Sloan-Kettering Institute, a Howard Hughes Medical Institute
investigator, a member of the National Academy of Sciences,
and the incumbent of the David H. Koch Chair in Immunologic
Studies at Memorial Sloan-Kettering. Dr. Allison is a leader in
the field of immunology, particularly in developing ways to
help the immune system recognize and destroy cancer cells.
His research is focused on the mechanisms that regulate the
immunological response of T lymphocytes, especially strategies to manipulate those responses in clinically relevant areas,
including autoimmunity, allergies, vaccinations, and tumor
therapy. Dr. Allison has shown that an immune-regulating
molecule called cytotoxic T lymphocyte-associated antigen-4
(CTLA-4) inhibits activated T-cells in the immune system and
prevents them from attacking the body’s own tissues. He and
his colleagues have now created antibodies to human CTLA-4
that are being studied in human clinical trials for the treatment
of melanoma, renal cell carcinoma, prostate cancer, and ovarian cancer.
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Tobias Raum, PhD, is head of the “Lead Generation group” of
Amgen Research Munich following the acquisition of Micromet
(Munich) by Amgen in early 2012. He had held the equivalent
position at Micromet since 2001. His group is involved in the
selection and optimization of human antibody fragments as a
starting point for novel antibody and BiTE programs. Several
candidate specificities selected by Dr. Raum’s group are currently in clinical trials or advanced preclinical phase. In addition, Dr. Raum also heads Amgen Research Munich’s “New BiTE
Platform group which is involved in the evaluation of BiTE antibodies against new indications. This group has recently demonstrated, that BiTE antibodies are able to efficiently mediate
eradication of cancer stem cells and that Tregs can be recruited
in BiTE antibody mediated killing of target cells. Prior to this,
Dr. Raum headed the Molecular Design group at Connex GmbH
(Munich) after having received his PhD from the University of
Munich in 1999.
Jay Berzofsky
National Cancer Institute
Bethesda, USA
Jay Berzofsky is Chief of the Vaccine Branch, Center for Cancer Research, National Cancer Institute. He joined NIH in
1974. Dr. Berzofsky’s research has focused on antigen processing and presentation by MHC molecules, the structure of
antigenic determinants, cytokine and regulatory cell control
of T-cell function and avidity, and translation to the design of
vaccines for AIDS, malaria, cancer, and viruses causing cancer. He has over 435 scientific publications and has received
a number of awards, including the U.S. Public Health Service
Superior Service Award, the 31st Michael Heidelberger Award,
the McLaughlin Visiting Professorship, the Australasian Society
for Immunology Visiting Lectureship, and the Tadeusz J. Wiktor Memorial Lectureship. He is past President of the American
Society for Clinical Investigation, and a Fellow of the American
Association for the Advancement of Science. He was also elected
Chair of the Medical Sciences Section of the American Association for the Advancement of Science (AAAS) 2007-08. He won
the NIH Director’s Award and NCI Merit Award in 2008.
Nicole Bidmon
TRON
Mainz, Germany
Nicole Bidmon is an immunologist who focuses on reference
samples to control immune assay performance for the most
commonly used cellular T-cell assays. She is currently working
in the laboratory for Cell Mediated Immunity (CMI) at TRON
gGmbH, Mainz and is a co-organizer of CIMT/CIP proficiency
panels to study the use of reference samples in T-cell assays
across institutions. She studied biotechnology at the University
of Applied Sciences, Berlin, and worked in a research group at
the Imperial College, London defining the structure and function of archaeal and eukaryotic RNA polymerases. At the Max
Delbrück Center for Molecular Medicine, Berlin, she completed
her diploma thesis studying a knock-out mouse model to understand muscle contraction regulation.
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CIMT | Speakers
CIMT | Speakers
2012 Speakers
2012 Speakers
Cedrik Britten
Thomas Gajewski
Cécile Gouttefangeas
Holger Hoff
Ribological GmbH
University of Chicago Medicine
Department of Immunology
TRON
Mainz, Germany
Chicago, USA
Tübingen, Germany
Mainz, Germany
Cedrik Britten is Vice President R&D at Ribological GmbH, a
Mainz-based biotech company developing innovative immunotherapy against cancer. In addition, Dr. Britten is the CIMT
Executive Director Translational Medicine and committee member of the CIMT Immunoguiding Program (CIP), where he is
involved in generating tools to enable technical validation of
T-cell assays across institutions and in promoting the concept
of assay harmonization in the context of immunological monitoring of immunotherapy trials. Dr. Britten is also an active
member of the CIMT Regulatory Research Group (RRG) that
strives to gain and maintain a deep understanding of regulatory principles and promotes a science-driven critical review
of existing regulatory documents in working out solutions to
regulatory challenges.
Peter Bross
FDA Center for Biological Evaluation
and Research (CBER)
Rockville, USA
Peter Bross is a clinical oncology team leader in the FDA Center
for Biological Evaluation and Research (CBER), and previously worked as a clinical reviewer in the Division of Oncology
Drug Products in the Center for Drug Evaluation and Research
(CDER). Dr. Bross has expertise in the design and analysis of
clinical oncology trials of cellular, tissue and gene therapies,
especially cancer vaccines, as well as in trial design, eligibility
criteria, dose escalation schema, definition of dose limiting toxicities, and study endpoints in relationship to decision making
for later phase trials. He is also involved in late phase clinical
oncology trials of biological therapies in terms of utilization of
comparator arms, primary endpoints and secondary endpoints.
Dr. Bross is a graduate of University of Virginia Medical School
and completed fellowship training in Hematology and Oncology
at The George Washington University. Dr. Bross has a particular
interest in companion diagnostics and personalized medicine.
Thomas Gajewski is a professor of pathology at the Department
of Medicine Section of Hematology/Oncology at the University
of Chicago Medicine, and the Ben May Institute. Dr. Gajewski
researches and develops new treatments for patients with melanoma, investigating the regulation of T-cell activation, T-cell
signaling, tumor immunology and immunotherapy of melanoma. His laboratory studies the molecular and cellular regulation of T lymphocyte activation and differentiation, and in turn
applies this information to preclinical and clinical efforts to
promote anti-tumor immunity in vivo. Dr. Gajewski serves as
an editor for Cancer Research. In addition he is the President of
the Society for Immunotherapy of Cancer (SITC). He has served
on the program committees for the American Society for Clinical Oncology (ASCO) and the American Association for Cancer
Research (AACR).
Jérôme Galon
INSERM
Paris, France
Jérôme Galon is Research Director for INSERM and Head of
the Integrative Cancer Immunology Laboratory at Cordeliers
Research Center in Paris. Dr. Galon has developed a multidisciplinary network with research scientists in immunology
and cancerology, clinical teams and bioinformaticians. Dr.
Galon believes that a global understanding of cancer requires
the integration and analysis of genomic, proteomic, transcriptomic, molecular, cellular, as well as clinical data and requires
bioinformatics. In 2010 he jointly received the William B. Coley
Award for groundbreaking studies demonstrating that the
“immune contexture” – including the functionality, location,
and density of immune infiltrate in colorectal tumors – is a
major prognostic factor for human cancers.
John Castle
University of Southampton
TRON
Southampton, UK
John Castle is an expert in computational biology, next-generation sequencing (NGS), and genomics. Originally trained in
physics at the University of Washington (Seattle) and at MIT,
Dr. Castle developed advanced inverse theory methodologies to
image earthquakes and plate tectonics. For the next ten years,
he worked at Rosetta Inpharmatics, later part of Merck Sharp
and Dohme (MSD), where he invented novel genomic technologies, including DNA microarrays, siRNAs, and NGS, and used
super computers to analyse and interpret the data to apply the
results to drug development. At TRON, he is Co-Director of the
Biomarker Development Center and combines immunology,
computational biology, and genomics using NGS to develop
individualized “on-demand” cancer vaccines.
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Martin Glennie is a Professor of Immunology and Head of the
Cancer Sciences Academic Unit at the University of Southampton, specializing in antibody effector systems. Throughout
his career Dr. Glennie has focused on translational research
and understanding and improving how monoclonal antibodies
(mAb) can be used in controlling cancer. Dr. Glennie has published more than 100 research articles in such peer-reviewed
journals as Nature, Nature Medicine, Journal of Experimental Medicine, etc. He is an adjunct Professor to the Dartmouth
Medical School, NH and consults widely for the biotech industry in Europe and the US. He is also a frequent reviewer for a
wide range of scientific journals and granting bodies, and sits
on review panels and advisory boards for Cancer Research UK
and the NIH.
Holger Hoff’s research is focused on the regulation of T-cell
activation and signal transduction. Dr. Hoff studied biology
at the University of Cologne and received his PhD at the German Arthritis Research Center in Berlin. After his post-doctoral
training at the Charité in Berlin, he moved to Mainz where he is
now Head of the “Designer T-cells” Functional Unit at TRON –
Translational Oncology at the University Medical Center Mainz.
His work aims at the generation of T-cells with customized characteristics and improved anti-tumoral potency for the application in adoptive T-cell therapies.
Sine Reker Hadrup
Patrick Hwu
Center for Cancer Immune Therapy,
University Hospital Herlev
University of Texas,
M. D. Anderson Cancer Center
Copenhagen Denmark
Houston, USA
Sine Reker Hadrup is Group Leader at the Center for Cancer
Immune Therapy, University Hospital Herlev, Copenhagen,
Denmark, and an external Associate Professor at the University
of Copenhagen, Institute of International Health, Immunology
and Microbiology. The main focus of Dr. Hadrup’s research
group is the use and development of high-throughput technologies for T-cell detection, the identification of new T-cell epitopes
and specific T-cell populations of relevance for cancer immune
therapy. Additionally, Dr. Hadrup has been involved in the
organization of the CIMT Immunoguiding Program (CIP) proficiency panels for MHC multimers for several years.
Wolfgang Herr
University Medical Center,
Johannes Gutenberg University, Mainz
Mainz, Germany
Martin Glennie
Mainz, Germany
Cécile Gouttefangeas is Group Leader at the Department of
Immunology in Tübingen, Germany. Her long-standing focus
is the characterization of adaptive immune responses against
cancer. This includes the description of tumor antigen-derived
T-cell epitopes, of antibodies recognizing tumor antigens and of
immune effector cells in cancer patients before or during immunotherapy. Dr. Gouttefangeas is also a founding member of the
international CIMT Immunoguiding Program (CIP) for harmonization of the techniques applied for monitoring antigen-specific
T-cells and is currently co-chairing the CIP steering committee.
Wolfgang Herr is Associate Professor of Medicine at the University Medical Center, Johannes Gutenberg University, Mainz,
Germany, where he specializes in hematology and medical
oncology. A post-doc research fellowship at the University of
Pittsburgh, USA, was followed by a bone marrow transplantation training program at the Fred Hutchinson Cancer Research
Center in Seattle, USA. Today, his major research interests
include T-cell depleted allogeneic HSCT protocols, integrating
adoptive immunotherapy with pathogen and leukemia-specific
T lymphocytes. Currently in charge of the Center’s clinical and
experimental hematopoietic stem cell transplantation (HSCT)
program, Dr. Herr also directs the clinical research group
KFO183 ‘Optimized Allogeneic Lymphocyte Therapy’ of the
Deutsche Forschungsgemeinschaft, which comprises several
research groups at the Universities of Mainz and Tübingen.
KFO183’s research focus is the development of immunomodulatory strategies to improve the efficiency and specificity of graftderived lymphocyte reactions in the context of allogeneic HSCT.
Patrick Hwu is Co-Director of the Center for Cancer Immunology Research and Chair of the Department of Melanoma Medical Oncology at the University of Texas, M. D. Anderson Cancer
Center with over 20 years of experience in the field of tumor
immunology and concept-to-clinic studies. He received training in Internal Medicine and Medical Oncology at the Johns
Hopkins Hospital and National Cancer Institute, respectively.
For over 10 years, he was a Senior Investigator at the National
Cancer Institute, and performed novel clinical and laboratory
studies of cancer immunotherapies. He has taken a number of
concepts from the laboratory to the clinic including studies of
vaccines and adoptive T-cell therapies. Throughout the years he
has trained numerous doctors, scientists, and medical students
who are currently pursuing careers in academic medicine.
Carl June
University of Pennsylvania, Abramson
Family Cancer Research Institute
Philadelphia, USA
Carl June is currently Director of Translational Research at the
University of Pennsylvania, and an Investigator of the Abramson Family Cancer Research Institute. Before his current position, Dr. June was Head of the Department of Immunology at
the Naval Medical Research Institute and Professor at the Uniformed Services University for the Health Sciences in Bethesda,
Maryland. He maintains a research laboratory that studies
various mechanisms of lymphocyte activation that relate to
immune tolerance and adoptive immunotherapy. Dr. June’s lab
has developed a large-scale tissue culture technique that permits the efficient propagation of polyclonal HIV CD4 and CD8
T-cell subsets. Several clinical trials involving adoptive immunotherapy of autologous and allogeneic T-cells are in process.
Four trials are using genetically engineered T-cells.
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CIMT | Speakers
CIMT | Speakers
2012 Speakers
2012 Speakers
Kyogo Itoh
Tibor Keler
Antonio Lanzavecchia
Michael Nishimura
Kurume University Medical School
Celldex Therapeutics
Institute for Research in Biomedicine
Loyola University
Kurume, Japan
Phillipsburg, USA
Bellinzona, Switzerland
Chicago, USA
Kyogo Itoh is Professor of Immunology at the Department of
Immunology and Immunotherapy at Kurume University Medical School. Dr. Itoh’s research is focused on the development of
personalized peptide vaccines (PPV), which involve a personalized selection of peptides suitable for each patient based on
pre-existing cellular and humoral responses, and vaccination of
patients with these selected peptides. He has conducted several
PPV trials in various cancer types, such as prostate cancers
and glioblastoma, showing feasibility and efficacy of the PPV
approach. Dr. Itoh also develops biomarkers for cancer vaccines
using novel tumor antigens and peptide-specific antibodies.
Sylvia Janetzki
ZellNet Consulting, Inc,
Assay Working Group
Fort Lee, USA
Sylvia Janetzki is the President of ZellNet Consulting, Inc., and
Coordinator of the Assay Working Group of the CIC/CRI. The
focus of her work has been immune monitoring approaches
for clinical studies. In 1998 she founded ZellNet Consulting, a
company specialized in the Elispot technique and its advancement. ZellNet cooperates with many organizations and institutions including the WHO and Elispot Resource Group, to
enhance the knowledge about and improve the performance of
Elispot assays. Her work also led to a tight collaboration with
the Cancer Immunotherapy Consortium (CIC/CRI), for which
she initiated and leads a proficiency panel program addressing
different assays like Elispot, Multimer staining, ICS, Luminex,
and others. This program, currently the largest program of its
kind involving more than 100 laboratories from around the
world, aims at a) offering an external validation program, and
b) enhancing assay harmonization. First assay harmonization
guidelines for the field have been published as a result of this
program.
Tibor Keler is currently Senior Vice President and Chief Scientific Officer of Celldex Therapeutics, a company he helped
found and spin out from Medarex in 2005. Dr. Keler had spent 12
years at Medarex overseeing research and pre-clinical development activities. Previously he was a post-doctoral fellow at Fox
Chase Cancer Center, and performed his graduate studies at the
University of Pennsylvania receiving his PhD in Microbiology
in 1989. His professional focus has been developing antibodybased therapeutics for treatment of cancer and infectious diseases.
Samir Khleif
Georgia Health Sciences University
Augusta, Georgia
Samir Khleif is the Director of Georgia Health Sciences University’s Cancer Center in Augusta, Georgia. Dr. Khleif is an
experienced researcher into how tumors manipulate and suppress immune response. He is a graduate of the University of
Jordan School of Medicine and was a Post-doctoral Research
Fellow at Michigan State University. Before his current position,
Dr. Khleif was Head of the Cancer Vaccine Section at the US
National Cancer Institute in Bethesda, Maryland. As a native of
Jordan, Dr. Khleif helped establish the King Hussein Institute
for Cancer and Biotechnology in Amman, Jordan. His research
focuses on integrating translational basic laboratory research
and clinical trials to understand the interaction between tumor
cells and the immune system and to develop cancer vaccines.
Robert Kralovics
Austrian Academy of Sciences
Vienna, Austria
Michael Kalos
University of Pennsylvania School
of Medicine
Philadelphia, USA
Michael Kalos is Adjunct Associate Professor in Pathology and
Laboratory Medicine, and Founding Director of the Translational and Correlative Studies laboratory at the University of
Pennsylvania School of Medicine where he is involved in the
translational/clinical development and biomarker evaluation of
novel cell-based immunotherapeutics. Dr. Kalos has authored
multiple primary and review articles as well as book chapters
in the field of cancer immunotherapy, and is a member of the
steering committees for the CIC/CRI immune monitoring working group and representative to the CIP immune monitoring
workgroup.
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Robert Kralovics is a Principal Investigator at the Research
Center for Molecular Medicine of the Austrian Academy of Sciences. Trained in biophysics, he did his post-doctoral work on
the genetics of myeloproliferative disorders working with Josef
Prchal at the University of Alabama at Birmingham, USA. He
followed Prchal as an Assistant Professor at the Baylor College
of Medicine in Houston. Robert Kralovics’ research interests
lie in chronic myeloproliferative disorders (MPDs). His major
achievement so far has been the identification of a gain-of-function mutation in the JAK2 kinase gene (V617F) that plays an
important role in MPD pathogenesis. Prominently published in
April 2005 in New England Journal of Medicine, the work has
given Robert instant celebrity in the hemato-oncological field
and fostered Robert’s interest in Jak2 as a potential therapeutic
target. One of his new research tasks is to find new mutations
using the latest genomics technologies.
Antonio Lanzavecchia is the Director of the Institute for
Research in Biomedicine in Bellinzona, Switzerland. He has
been Professor of Immunology at the University of Genoa and at
the University of Siena. Dr. Lanzavecchia’s research has covered
several aspects of human immunology: antigen processing and
presentation, dendritic cell biology, lymphocyte activation and
traffic and, more recently, the cellular basis of T and B cell memory and the production of human monoclonal antibodies. He
was awarded the EMBO medal in 1988 and the Cloëtta prize for
deciphering the signals that regulate T-cell mediated immunity.
In 2009, Dr. Lanzavecchia was named Full Professor of Immunology at the ETH in Zurich. Dr. Lanzavecchia has published
more than 200 papers. His research has covered several aspects
of human immunology: antigen processing and presentation,
dendritic cell biology, lymphocyte activation and traffic, T and
B cell memory. Recently he developed a method for the efficient
isolation of human monoclonal antibodies from memory B cells,
which has been successfully applied to infectious diseases such
as SARSCoV, H5N1, HCMV, Dengue, Malaria and HIV-1.
Michael Nishimura is a Professor in the Department of Surgery
at Loyola University Chicago. He is also Associate Director of
the Oncology Institute, Associate Director of the Cancer Center
Translations Research, and Program Director of Immunologic
Therapeutics at Loyola. His laboratory has had a long standing
interest in the genetics of T-cell receptor (TCR) genes that mediate recognition of tumor and viral antigens. To improve the therapeutic efficacy of TCR gene modified T-cells, Dr. Nishimura’s
laboratory has several ongoing projects designed to understand
the biology of TCR transduced T-cells. Using a combination of
mouse in vivo tumor models, in vitro human studies, and clinical trials, his laboratory is studying the mechanisms to increase
the persistence and function of adoptively transferred T-cells.
Another critical problem his laboratory is addressing is how to
circumvent tumor immune escape. And finally, it is developing
novel approaches for generating TCR transduced T-cells to treat
cancer.
Rienk Offringa
Ignacio Melero
German Cancer Research Center (DKFZ)
University of Navarra
Heidelberg, Germany
Navarra, Spain
Ignacio Melero serves as a Full Professor of Immunology at the
University of Navarra (CIMA and Clínica Universitaria), where
he leads a group on translational tumor immunotherapy with
emphasis on cell therapy, cytokine gene therapy and immunostimulatory mAbs. He earned a PhD in immunology working
on Natural Killer cell receptors with Miguel López-Botet. His
work contributed to the functional identification of the KIRs,
surface receptors that are key for the function of Natural Killer
cells and subsets of T-cells. Dr. Melero’s post-doctoral experience in the USA was geared towards experimental tumor immunology and understanding co-stimulation and co-inhibition in
the cellular immune response against cancer. His work contributed to the definition of immunological ignorance of tumor
antiges and, more importantly, contributed decisively to T-cell
co-stimulation via CD137. A main focus of his current research
are immunotherapy clinical trials involving cell therapy and
immunostimulatory monoclonal antibodies. Dr. Melero has
been awarded the BIAL Prize of Medicine (Portugal, 2005) and
other honors.
Rienk Offringa is Head of the Molecular Oncology of Gastrointestinal Cancers Division at the German Cancer Research Center
(DKFZ) and of the Pancreas Carcinoma Research Division at
the Surgery Clinic of Heidelberg University. His research aims
at developing immunotherapeutic strategies for modulation of
endogenous T-cell immunity in patients with resectable pancreatic cancer. For this group of patients, Dr. Offringa also focuses
on routine exome sequencing and mRNA expression profiling
and development of tools for functional studies, particularly
concerning tumor-stroma interaction in human pancreatic ductal adenocarcinoma.
Christian Ottensmeier
University of Southampton
Southampton, UK
Christian Ottensmeier is Professor in Experimental Cancer Medicine at the University of Southampton and Leader of the Southampton Experimental Cancer Medicine Centre. He has been a
consultant in medical oncology since 2000, with a clinical focus
on thoracic malignancies and melanoma. He co-developed a
number of national NCRI studies in lung cancer and manages
a broad and active clinical trials portfolio in both lung cancer
and melanoma. His laboratory group focuses on the preclinical development and early-phase clinical testing of strategies
to induce anti-tumour immune responses in patients. After an
initial focus on B-cell malignancies, the majority of his work
focuses on solid tumors. This has led to three linked, but distinct areas of investigation: detailed immunological evaluation
of the effect of immunological intervention in patients, assay
development and validation, and mechanistic studies in murine
models as well as humans of immune responses to vaccination
for further preclinical cancer vaccine development. The aim is
to complete the loop back into the clinic.
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CIMT | Speakers
CIMT | Speakers
2012 Speakers
2012 Speakers
Klaus Pantel
Hansjörg Schild
Pramod Srivastava
Scott D. Tanner
University Medical Center
Hamburg-Eppendorf
Johannes Gutenberg University Mainz
University of Connecticut School
of Medicine
DVS Sciences, Inc.
Hamburg, Germany
Klaus Pantel is Professor and Chairman of the Institute of Tumor
Biology at the University Medical Center Hamburg-Eppendorf.
The institute is part of the Hubertus Wald Cancer Center/University Cancer Center Hamburg (UCCH). After his post-doctoral
period in the USA on hematopoietic stem cell regulation (Wayne
State University, Detroit), he performed research on cancer
micrometastasis at the Institute of Immunology, University of
Munich for 10 years. The pioneer work of Dr. Pantel in the field
of cancer micrometastasis and circulating tumor cells is reflected by more than 250 publications in excellent high-ranking
biomedical and scientific journals and numerous awards (e.g.
AACR Outstanding Investigator Award 2010, German Cancer
Award 2010, ERC Advanced Investigator Grant 2011). Moreover,
Dr. Pantel was co-ordinator of the FP6 EU STREP “DISMAL”
(Disseminated Malignancies); he serves on the Editorial Boards
of international cancer journals (e.g. Clin. Cancer Res., Breast
Cancer Res.) and organizes international symposia (ISMRC) on
minimal residual cancer and circulating tumor cells.
Ugur Sahin
TRON
Mainz, Germany
Ugur Sahin is the founder and Managing Director of Science and
Research at TRON (Translational Oncology at Mainz University
Medical Center) where he is working towards developing novel
cancer vaccines and individualized cancer immunotherapies.
His current research focuses on the identification and characterization of new tumor antigens, the development of RNAbased cancer vaccines, and the development of diagnostic tools
for the early detection of cancer. Dr. Sahin holds several patents on novel, highly tumor-specific tumor antigens as well as
on RNA technology to achieve design and synthesis of highly
potent RNA molecules for induction of immunity. In addition to
being at the helm of TRON, Dr. Sahin serves as CEO of BioNTech
and leads an independent oncology research team at Johannes
Gutenberg University Mainz.
Mainz, Germany
Hansjörg Schild is Professor of Immunology and Managing
Director of the Immunology Research Center at Johannes Gutenberg University in Mainz. Dr. Schild graduated in human biology. He was a post-doctoral fellow at Stanford University Medical
School, Group Leader of the Department of Tumorvirus-Immunology of the German Cancer Research Center in Heidelberg and
Group Leader at the Department of Immunology at the University of Tübingen. Dr. Schild is an expert in the immunobiology
of heat shock proteins, adjuvants and antigen processing and
presentation. In 2000 he received the Georges Köhler Award of
the German Society for Immunology.
Robert Schreiber
Washington University School of Medicine
St. Louis, USA
Robert Schreiber is Professor of Molecular Microbiology at the
Washington University School of Medicine in St. Louis, USA. Dr.
Schreiber was the first to show that an immune system protein,
interferon gamma, activates immune cells called macrophages,
making it possible for them to attack microbial invaders and
tumor cells. His lab worked out many of the key aspects of
this activation process, including detailing the structure of the
interferon gamma receptor on macrophages and identifying the
signaling proteins activated by the receptor. Dr. Schreiber and
his colleagues also have proposed and won wide acceptance
for a new theory of immune cell and cancer cell interaction
called cancer immunoediting and produced the first experimental evidence for tumor eradication and tumor equilibrium. His
current work is aimed at defining interferon-gamma specific
roles in the tumor surveillance process, elucidating the genetic
mechanisms employed by tumors to become interferon-gamma
insensitive and exploring the relationships between interferongamma sensitivity of a tumor and its growth aggressiveness
in vivo.
Mark Sliwkowski
Richard H. Scheuermann
Genentech, Inc.
UT Southwestern Medical Center
San Francisco, USA
Dallas, USA
Richard H. Scheuermann is Chief of the Division of Biomedical
Informatics and holder of the John H. Childers Professorship
in Pathology at UT Southwestern Medical Center (Dallas). His
recent research focus has been in the development of new algorithms for the analysis of gene expression microarray, genetic,
comparative genomics, flow cytometry and biological network
data, and in the development of approaches and standards for
biomedical knowledge representation in the areas of immunology and infectious diseases research. He leads three large database development projects funded by the U.S. National Institutes of Health – the Influenza Research Database, the Virus
Pathogen Bioinformatics Resource Center and the Immunology
Database and Analysis Portal, and also serves as the Biomedical
Informatics Key Function Lead for UT Southwestern’s Clinical
and Translational Science Award (CTSA).
16
Mark Sliwkowski is currently a Senior Staff Scientist in Research
Oncology at Genentech, Inc. He received his B.S. from the University of Delaware and his PhD in Biochemistry from North
Carolina State University. Mark was a post-doctoral fellow in the
laboratory of Dr. Theresa C. Stadtman in the Laboratory of Biochemistry, in the National Heart, Lung, and Blood Institute at
NIH, where he studied bacterial enzymes that require selenium
for their catalytic activity. Upon leaving NIH, he joined Triton
Biosciences, Inc., where he studied growth factor receptors and
their ligands. Mark joined Genentech in 1991 as a Senior Scientist and worked on a number of programs involving drugs
directed against the human epidermal growth factor receptor
family (also known as the HER or ErbB family). Two of these
drugs, Herceptin® (trastuzumab) and Tarceva® (erlotinib) have
received U.S. Food and Drug Administration approval.
Farmington, USA
Pramod Srivastava is Professor of Immunology and Medicine,
and Director of the Carole and Ray Neag Comprehensive Cancer Center at the University of Connecticut School of Medicine.
Dr. Srivastava’s contributions lie in the areas of the immunological functions of heat shock proteins and cancer immunology. The heat shock protein based cancer vaccine, vitespen or
Oncophage, which was the first therapeutic cancer vaccine to
be approved for clinical use anywhere in the world, was based
on his discoveries, and was developed by him. After having
obtained degrees in biology and chemistry as well as botany
(paleontology), he studied yeast genetics and completed his
PhD in Biochemistry at the Center for Cellular and Molecular
Biology, Hyderabad, India. He also trained at Yale University
and Sloan-Kettering Institute for Cancer Research. Dr. Srivastava obtained his MD degree from the University of Connecticut
School of Medicine. He is widely published in scholarly journals
and serves on editorial boards for several journals in immunology. He is an inventor on about a hundred awarded patents, and
co-founded a number of biotechnology companies.
Renata Stripecke
Hanover, Medical School
Hanover, Germany
Renata Stripecke was trained in molecular biology, hematopoietic gene therapy and immunotherapy. At the University
of Southern California and the University of California at Los
Angeles, she held professorships in the field of genetic programming of dendritic cells. Since 2007, Dr. Stripecke has been
heading the Lymphatic Cell Therapy Program within the Excellence Cluster Rebirth and has been an Associate Professor at
the Department of Hematology at Hanover Medical School. Her
research topic is the translation of genetic reprogramming of
dendritic cell (DC) precursors into effective cell vaccines tailored for immune regeneration. DCs can be genetically programmed with designed lentiviral vectors in order to induce
differentiation of highly viable and potent antigen-presenting
cells. These lentivirus-induced DCs (iDCs) can be produced
under clinically compliant conditions in just one day of ex vivo
culture, which drastically simplifies clinical development. The
R&D projects are aimed at immunotherapy of melanoma and
leukemia and cytomegalovirus after stem cell transplantation.
Markham, Canada
Scott D. Tanner is co-founder and Chief Technology Officer of
DVS Sciences, Inc., a biotech company that develops, manufactures and markets analytical instruments and reagents for high
throughput, massively multi-parameter single cell analysis. He
is also a Professor in the Department of Chemistry at the University of Toronto, Canada. Dr. Tanner received the University
of Toronto 2011 Inventor of the Year Award in Biomedical and
Life Sciences, the 2011 ThermoFisher Scientific Spectroscopy
Award, the 2003 W.A.E. McBryde Medal, and the 2001 Manning
Innovation Foundation Award of Distinction. He is a Fellow of
the Royal Society of Chemistry (UK) and sits on the Editorial
Board of the RSC’s Journal of Analytical Atomic Spectrometry.
Zlatko Trajanoski
Innsbruck Medical University
Innsbruck, Austria
Zlatko Trajanoski is Professor and Head of Bioinformatics at
Innsbruck Medical University. Dr. Trajanoski focuses on the
development of methods for the integration of large-scale biomolecular (transcriptomic, proteomic) and clinical data, and their
application to complex diseases. For clinical applications, Dr.
Trajanoski aims to integrate disparate data sources, including
genomic sequences, gene expression data, proteomic data, clinical data, and patient-related information, and to enable queries
and analyses across disparate data sources. Using mathematical
modeling approaches, Dr. Trajanoski addresses biological questions in cell differentiation and cancer, developing multi-scale
models on tumor-immune cell interaction (i.e. models at the
subcellular, cellular, and tissue level) to reveal immune signals
controlling tumor progression.
Sjoerd H. van der Burg
Leiden University Medical Center
Leiden, Netherlands
Sjoerd H. van der Burg leads the experimental cancer immunology and therapy group with about 20 scientists and technicians
at the Department of Clinical Oncology at Leiden University
Medical Center. Dr. van der Burg’s laboratory focuses on immunomonitoring and immunotherapy of solid tumors. His special
research interest is in experimental cancer immunology with
an emphasis on local immune response, T-helper cells, regulatory T-cells and cytotoxic T-cells in tumor immunity, as well
as in cancer immunotherapy, in particular the development of
therapeutic vaccine strategies and adoptive transfer of ex-vivo
expanded T-cells. Dr. van der Burg has extensive experience in
monitoring human T-cell responses and uses this experience to
guide the development of therapeutic vaccines against cancer.
He has both initiated and participated in 10 different clinical
trials aiming at cancer immunotherapy over the last decade.
At present, he co-chairs the CIMT Immunoguiding Program
(CIP) which focuses on the harmonization of T-cell assays in
Europe. Dr. van der Burg has already published over 100 original research papers on these topics.
17
CIMT | Speakers
CIMT | Program Schedule
2012 Speakers
Robert H. Vonderheide
Laurence Zitvogel
Perelman School of Medicine
Institut National de la Santé et
Recherche Médicale
Philadelphia, USA
Robert H. Vonderheide is Associate Professor at the Perelman
School of Medicine at the University of Pennsylvania and Associate Investigator at the Abramson Family Cancer Research
Institute. He is Associate Director for Translational Research
at the Abramson Cancer Center. Dr. Vonderheide’s laboratory
combines efforts in both basic research and clinical investigation to advance the understanding of tumor immunology and
develop novel immunotherapies for cancer. His basic research
includes deciphering the immunobiology of a novel genetically
engineered mouse model of pancreatic cancer, including the
regulation of immune surveillance and the tumor microenvironment by CD40 and other inflammatory pathways. His translational work tests novel vaccines and antibody approaches for
the treatment of patients with pancreatic cancer, melanoma,
and breast cancer. He has studied telomerase and survivin as
tumor rejection antigens, and the immune modulatory capacity
of CD40, CTLA-4, and CD25 antibodies.
Steffen Walter
Program Schedule CIMT 2012
May 23
Day 1
Time:
Location:
Program:
08:30-9:00
Foyer
Welcome Coffee
09:00-9:30
Hall A
Welcome Address
Paris, France
Laurence Zitvogel is Research Director at Institut National de
la Santé et Recherche Médicale U1015, in a laboratory located at Institut Gustave Roussy, and the Head of the Center for
Clinical Investigations CICBT 507 for vaccine developments at
Villejuif, France. She started her scientific career at the University of Pittsburgh, USA, in Michael Lotze’s laboratory. Dr.
Zitvogel actively contributed to the field of cancer immunology and immunotherapy, and she brought together basic and
translational research, including the design of cancer therapies
through combined animal studies and Phase I patient trials. Her
expertise is mainly in dendritic cell and innate effector biology
and relevance during tumor development as well as exosomebased vaccine designs. She pioneered the concept of immunogenic cell death and showed that chemotherapy, radiotherapy
and inhibitors of tyrosine kinase mediate their tumoricidal
activity, at least partly through the immune system.
09:30-10:00
Hall A
Christoph Huber (CIMT Chairman, Mainz)
Doris Ahnen (Rhineland-Palatinate Secretary of Education, Science,
Further Education and Culture, Mainz)
Opening Lecture
Chair: Thomas Wölfel (Mainz, Germany)
Pramod K. Srivastava University of Connecticut School of Medicine
(Farmington, USA)
Cancer immunology: search for specificity
Immatics Biotechnologies
Tübingen, Germany
Steffen Walter is Director and Head of Immunology at Immatics Biotechnologies. Dr. Walter is in charge of the most relevant immunological data (immunomonitoring) from immatics’ phase I-III clinical vaccination trials that allow optimal
informed decisions for drug development. This includes the
setup of one of the largest international laboratory networks
to collect high-quality PBMC samples from multi-centric clinical trials, assessment of T-cell specificity and function as well
as state-of-the-art measurement of many cellular biomarkers to
define the immune status of a patient. Furthermore, his team
pre-clinically assesses the immunogenicity of novel product
candidates. Dr. Walter supports and participates in the work of
the immunomonitoring community (CIMT-CIP, CIC) that aims
to establish urgently needed standardization and harmonization to allow immunomonitoring become a fully accepted clinical trial endpoint.
10:00-12:00
Plenary Session 1 / Tumor Vaccination
Chairs: Cedrik Britten (Mainz, Germany), James P. Allison (New York, USA)
10:00-10:30
Tibor Keler Celldex Therapeutic, Inc. (Phillipsburg, USA)
Approaches to improving anti-tumor immunity
10:30-11:00
Jay Berzofsky National Cancer Institute – Center for Cancer Research
(Bethesda, USA)
Therapeutic T-cell epitope cancer vaccines: blockade of negative regulation
to enhance vaccine efficacy.
11.00-11:30
James P. Allison Memorial Sloan Kettering Cancer Center (New York, USA)
Immune checkpoint blockade: New insights and opportunities
11.30-12:00
Robert Schreiber Washington University School of Medicine (St. Louis, USA)
Deconstructing cancer immunoediting
12:00-13:30
18
Hall A
Foyer
Lunch Break
19
CIMT | Program Schedule
CIMT | Program Schedule
Program Schedule CIMT 2012
May 23
Day 1
Program Schedule CIMT 2012
May 23
Day 1
Time:
Location:
Program:
Time:
Location:
Program:
12:00-13:30
Hall B
CIP Breakout Session
16:30-17:10
Hall A
Feature Lecture
Chairs: Cédrik Britten (Mainz, Germany), Cécile Gouttefangeas (Tübingen, Germany)
Chair: Carl Figdor (Nijmegen, Netherlands)
Jérôme Galon INSERM (Paris, France)
The Immunoscore: A new approach for the classification of cancer in
the era of immunotherapy
Thomas Gajewski Society for Immunotherapy of Cancer (Milwaukee, USA)
Innate and adaptive immunity within the tumor microenvironment
Cécile Gouttefangeas University of Tübingen (Tübingen, Germany)
CIP activities 2011-2012
Sylvia Janetzki Zellnet Consulting, Inc. (Fort Lee, USA)
MIATA – introduction of the final guidelines for structured reporting of results
from T-cell assays
Sine Hadrup Center for Cancer Therapy (Herlev, Denmark)
1st results from the new CIP MHC multimer panel – evaluation of different
fluorochromes for MHC multimer staining
Nicole Bidmon University Medical Center Mainz (Mainz, Germany)
Reference samples to control T-cell assay performance
Christian Ottensmeier University of Southampton (Southampton, UK)
In-vitro culture of T-cells
Steffen Walter Immatics Biotechnologies GmbH (Tübingen, Germany)
Objectives of a MDSC proficiency panel
13:30-15:00
Hall A
Hall A
Late Breakout Session / Immunomics
Chairs: Bernhard Korn (Mainz, Germany), Michael Koslowski (Biberach, Germany)
17:15 -17:45
Robert Kralovics CeMM Research Center for Molecular Medicine (Vienna, Austria)
Genetic heterogeneity of myeloproliferative disorders and implications for therapy
17:45-18.15
Richard Scheuermann UT Southwestern Medical Center (Dallas, USA)
Toward automated analysis of flow cytometry data
18:15 -18:45
Zlatko Trajanoski Innsbruck Medical University (Innsbruck, Austria)
Immune score for stratification of patients with colorectal cancer
18:45-19:15
John Castle TRON (Mainz, Germany)
Exploiting the mutanome for tumor vaccination
17:30-19:00
Hall C
CIMT Members Meeting
Plenary Session 2 / Individualized Medicine
Chairs: Hans-Georg Rammensee (Tübingen, Germany), Hyam Levitsky (Nutley, USA)
13:30-14:00
Klaus Pantel University Medical Center Hamburg-Eppendorf
(Hamburg, Germany)
Circulating tumor cells as biomarkers in cancer patients
14:00-14:30
Laurence Zitvogel INSERM (Villejuif, France)
The anti-cancer immune response – indispensable for therapeutic success?
14:30-15:00
Kyogo Itoh University School of Medicine (Kurume, Japan)
Personalized peptide vaccination for advanced cancer patients
15:00-16:30
Foyer
Coffee Break
15:00-16:30
Foyer
Poster Session I
20
17.15 -19.15
21
CIMT | Program Schedule
CIMT | Program Schedule
Program Schedule CIMT 2012
May 24
Day 2
Program Schedule CIMT 2012
May 24
Day 2
Time:
Location:
Program:
Time:
Location:
Program:
08:30-10:00
Hall A
Plenary Session 3 / Antibodies
10:30-12:00
Hall B
Short Talk Session II / Improving Immunity
Chairs: Özlem Türeci (Mainz, Germany), Martin Glennie (Southampton, UK)
Chairs: Thomas Gajewski (Chicago, USA), Robert Vonderheide (Philadelphia, USA)
08:30-9:00
Martin Glennie University of Southampton (Southampton, UK) Optimizing agonistic antibodies to promote anti-cancer responses
10:30-10:45
Patrice R. Douillard (#044) Baxter Innovations GmbH, (Vienna, Austria) Immunotherapeutic potential of fully human anti-macrophage migration
inhibitory factor antibodies in different mouse cancer models
09:00-9:30
Tobias Raum Amgen Research (Munich, Germany) How BiTE antibodies can change the game in oncology
10:45-11:00
Laurent Derré (#052) Lausanne University Hospital (CHUV) (Lausanne, Switzerland) CpG-ODN induced upregulation of BTLA mediating selective inhibition of human B cells
09:30-10:00
Mark Sliwkowski Genentech (San Francisco, USA) Multiple approaches to target HER/ErbB receptors in solid tumors
11:00-11:15
Andreas Lundqvist (#060) Karolinska Institutet (Stockholm, Sweden) Zoledronic acid-treated monocytes augment TRAIL-mediated cytotoxicity of human NK cells
11:15 -11:30
Sissela Broos (#063) Lund University (Lund, Sweden) Synergistic augmentation of CD40-mediated activation of antigen-presenting
cells by amphiphilic poly(γ-glutamic acid) nanoparticles
11:30-11:45
C. Di Donna (#073) Regina Elena National Cancer Institute (Rome, Italy) Effective chemo/immunotherapy in melanoma patients activates non-canonical AKT
+
pathway of Melan-A+ tumor-specific CD8 T-cell clones
11:45 -12:00
Mustafa Diken (#079) TRON (Mainz, Germany) mTOR inhibitor Rapamycin enhances the cancer therapeutic potency of naked RNA vaccine
10:00-10:30
North Foyer
Coffee Break
10:30-12:00
Hall A
Short Talk Session I / Cellular Therapy
Chairs: Philipp Beckhove (Heidelberg, Germany), Udo Hartwig (Mainz, Germany)
10:30-10:45
Jenny J. Hong (#002) National Cancer Institute, NIH (Bethesda, USA) Successful treatment of melanoma brain metastases with adoptive cell therapy
10:45-11:00
Cordula Gründer (#003) UMC Utrecht (Utrecht, Netherlands) γ9- and δ2-CDR3 domains regulate functional avidity of T-cells harbouring
γ9δ2T-cell receptors
11:00-11:15
Congcong Zhang (#006) Georg-Speyer-Haus (Frankfurt, Germany) Retargeted natural killer cells for adoptive immunotherapy of glioblastoma
11:15 -11:30
Andrea Bloetz (#013) University Medical Center of Johannes Gutenberg University
(Mainz, Germany) +
Targeting leukemia using allo-HLA-DQ and allo-HLA-DP specific CD4 T-cells
11:30-11:45
Sabine Mall (#021) Klinikum rechts der Isar at Technical University (Munich, Germany) Development of clinically implementable imaging strategies for T-cell
receptor-transgenic T-cells
11:45 -12:00
Christian Krug (#042) Universitätsklinikum Erlangen (Erlangen, Germany) Generation of MCSP-specific, MHC-independent T-cells by RNA electroporation at
a clinically feasible scale
22
10:30-12:00
Hall C
Short Talk Session III / Immunomonitoring
Chairs: Sylvia Janetzki (Fort Lee, USA), Marij Welters (Leiden, USA)
10:30-10:45
Anna-Lena Krause (#088) German Cancer Research Center (DKFZ) (Heidelberg, Germany) T-cell recognition of tumor-associated antigens in patients with preinvasive lesions
of the breast
10:45 -11:00
Pia Kvistborg (#096) Netherlands Cancer Institute (Amsterdam, Netherlands) Dissection of anti-CTLA4-induced cytotoxic T-cell responses in melanoma
11:00-11:15
Cindy Desmarais (#097) Adaptive Biotechnologies (Seattle, USA) Tracking T-cell clones using high-throughput sequencing of antigen receptor CDR3 chains
11:15 -11:30
Henning Zelba (#100) University Hospital Tübingen (Tübingen, Germany) NY-ESO-1 and Melan-A-reactive T-cells are predictive for the clinical outcome
of late-stage melanoma patients
11:30-11:45
Craig Slingluff (#104) University of Virginia (Charlottesville, USA) Activation, dysfunction and retention of antigen-reactive T-cells in the vaccine site
microenvironment after multipeptide vaccine in incomplete Freund’s Adjuvant
11:45 -12:00
Steffen Walter (#107) immatics biotechnologies (Tübingen, Germany) IMA910, a novel multi-peptide cancer vaccine for advanced colorectal cancer, induces
+
+
multiple CD8 and CD4 T-cell responses associated with improved survival
23
CIMT | Program Schedule
CIMT | Program Schedule
Program Schedule CIMT 2012
May 24
Day 2
Program Schedule CIMT 2012
May 24
Day 2
Time:
Location:
Program:
Time:
Location:
Program:
12:00 - 14:00
North Foyer
Lunch Break
17:30 - 18:30
Hall A
Keynote Lecture
Chair: Christoph Huber (Mainz, Germany)
12:30 - 14:00
Hall B
Industry Satellite Symposium I
Antonio Lanzavecchia Institute for Research in Biomedicine (Bellinzona,
Switzerland)
Dissecting the human immune response to pathogens
Innovative Strategies for Personalized Cellular Immunotherapy
(organized by Miltenyi Biotec)
Ignacio Melero, University of Navarra (Pamplona, Spain)
Type I dendritic cells in translational research
19:30 - 24:00
A; West and
Social Event with Poster Award Ceremony
South Foyer
sponsored by
Renata Stripecke, Hanover Medical School (Hanover, Germany)
Next-generation dendritic cells programmed from inside out with lentiviral
vectors and clinical perspectives
Tobias Feuchtinger, University Children’s Hospital of Tübingen
(Tübingen, Germany)
+
+
Polyfunctional CD4 and CD8 T-cell grafts for adoptive T-cell transfer
against tumor antigens
14:00 - 16:00
Hall A
with a special performance by
The Checkpoints
Plenary Session 4 / Cellular Therapies
Chairs: Pedro Romero (Lausanne, Switzerland), Matthias Theobald (Mainz, Germany)
14:00 - 14:30
Carl June University of Pennsylvania (Philadelphia, USA)
Updates with CAR T-cells
14:30 - 15:00
Patrick Hwu MD Anderson Cancer Center (Houston, USA)
Rational combinations of targeted therapy and immunotherapy
15:00 - 15:30
Michael Nishimura Loyola University Medical Center (Maywood, USA)
TCR gene modified T-cells for adoptive immunotherapy
15:30 - 16:00
Wolfgang Herr University Medical Center Mainz (Mainz, Germany)
Toward specific allogeneic T-cell therapy in acute leukemia
16:00 - 17:30
North Foyer
Coffee Break
16:00 - 17:30
East Foyer
Poster Session II
24
25
CIMT | Program Schedule
CIMT | Program Schedule
Program Schedule CIMT 2012
May 25
Day 3
Program Schedule CIMT 2012
May 25
Day 3
Time:
Location:
Program:
Time:
Location:
Program:
08:30-10:30
Hall A
Plenary Session 5 / Improving Immunity
11:00-12:15
Hall B
Chairs: Ugur Sahin (Mainz, Germany), Cornelius Melief (Leiden, Netherlands)
Short Talk Session IV / New Targets,
Leads and Biomarkers
08:30-09:00
Shizuo Akira Osaka University (Osaka, Japan) Innate immune responses
Chairs: Steffen Walter (Tübingen, Germany), Nadeem Sheikh (Seattle, USA)
09:00-09:30
Sjoerd van der Burg Leiden University Medical Center (Leiden, Netherlands) Evasion of protective HPV-specific T-cell responses
11:00-11:15
Sandra Höfflin (#117) Universitätsklinikum Erlangen (Erlangen, Germany) Targeting mutated and overexpressed tumor antigens in cancer immunotherapy
09:30-10:00
Hansjörg Schild FZI - Research Center Immunology (Mainz, Germany) Mechanisms controlling adaptive immune responses
11:15 -11:30
Sabrina Prommersberger (#118) Universitätsklinikum Erlangen (Erlangen, Germany) Mutated BRAF and NRAS proteins as possible targets for the immunotherapy of melanoma
10:00-10:30
Rienk Offringa German Cancer Research Center (Heidelberg, Germany) Evaluation of therapeutic strategies in a genetically engineered mouse model for melanoma
11:30-11:45
Jochen Greiner (#131) University of Ulm (Ulm, Germany) Epitopes derived from the mutated region of Nucleophosmine 1 (NPM1) induce both
+
+
CD4 and CD8 T-cell responses
Coffee Break
11:45 -12:00
Richard Buka (#136) University of Birmingham (Birmingham UK) Lymphoma-specific immunity in healthy individuals and patients targets
phosphorylated antigens derived from the cytoplasmic tail of CD19
Plenary Session 6 / Regulatory Research
12:00-12:15
Karina Silina (#139) Latvian Biomedical Research and Study Centre (Riga, Latvia) Identification of a tumour-associated autoantibody signature with high diagnostic
value for early detection of gastric cancer
10:30-11:00
11:00-13:30
North Foyer
Hall A
Chairs: Harpreet Singh (Tübingen, Germany), Ulrich Kalinke (Hanover, Germany)
11:00-11:05
Ulrich Kalinke Twincore (Hanover, Germany) Introduction
11:05-11:30
Cedrik Britten Ribological (Mainz, Germany) RRG classification and regulatory path for APVACs – progress report
11:30-11:55
Ugur Sahin TRON (Mainz, Germany) Exploiting the mutanome for cancer vaccination
12:00-12:30
Samir Khleif NIH (Bethesda, USA) Vaccines targeting mutations in known antigens
12:30-13:00
Peter Bross Food and Drug Administration (Rockville, USA) US regulatory considerations for the development of therapeutic cancer vaccines:
an FDA reviewer's perspective
13:00-13:30
Bruno Flamion University of Namur (Namur, Belgium) The EMA approach to personalized medicine: Does it make a difference or should
payers decide?
26
11:00-12:15
Hall C
Short Talk Session V / Therapeutic Vaccination
Chairs: Sebastian Kreiter (Mainz, Germany), Kyogo Itoh (Kurume, Japan)
11:00-11:15
Lukasz Bialkowski (#156) Vrije Universiteit Brussel (Brussels, Belgium) Targeting cancer stem cells by raising cytotoxic T-cell responses against the stemness
protein SOX2
11:15 -11:30
Karl-Josef Kallen (#162) CureVac GmbH (Tübingen, Germany) Intradermal immunization with a novel mRNA-based vaccination technology induces
strong T- and B-cell responses in phase I/IIa trials in non-small-cell lung cancer (NSCLC)
and prostate carcinoma (PCA)
11:30-11:45
Stefanie Mandl (#170) BN Immuno Therapeutics (Mountain View, USA) Active immunotherapy of cancer with PROSTVAC® and MVA-BN®-HER2 demonstrate
potent preclinical anti-tumor efficacy
11:45 -12:00
Gennaro Ciliberto (#173) IRCCS Istituto Nazionale Tumori Fondazione G. Pascale
(Naples, Italy) A novel minigene scaffold for cancer vaccine applications
12:00-12:15
Gal Cafri (#175) Weizmann Institute of Science (Rehovot, Israel)
Tumor immunity conferred by mRNA-transfected dendritic cells expressing
bi-functional polypeptides which couple MHC-I presentation to dendritic cell activation
27
CIMT | Program Schedule
CIMT | Program Schedule
Program Schedule CIMT 2012
May 25
Day 3
Program Schedule CIMT 2012
May 25
Day 3
Time:
Location:
Program:
Time:
Location:
Program:
12:15-13:30
Hall B
Short Talk Session VI / Personalized Medicine
13:30-15:00
North Foyer
Lunch Break
13:30-15:00
Hall C
Industry Satellite Symposium II
Chairs: Stefan Stevanovic (Tübingen, Germany), Sjoerd van der Burg
(Leiden, Netherlands)
12:15-12:30
Else Marit Inderberg Suso (#022) Oslo University Hospital-Norwegian Radium
Hospital (Oslo, Norway) Adoptive transfer of redirected T-cells targeting a TGFβRII frameshift mutation
frequently occuring in microsatellite instable colon cancer
12:30-12.45
Tana Omokoko (#039) TRON (Mainz, Germany) Next-generation sequencing of γδ T-cell receptor repertoires
12.45-13:00
Satoko Matsueda (#089) Kurume University School of Medicine (Kurume, Japan)
Antibodies against CTL epitopes from tumor-associated antigens were widely
detectable in humans: potential prognostic significance in cancer patients
13:00-13:15
Markus Löffler (#151) University of Tübingen (Tübingen, Germany)
Actively personalized multi-peptide vaccination for primary
liver cancers – a novel strategy for overcoming residual disease
13:15 -13:30
Jean-Paul Rivals (#196) University Hospital (Lausanne, Switzerland) Correlation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with
clinical and pathological relationships of patients with oral squamous cell carcinoma
Short Talk Session VII / Tumor Biology and
Interaction with the Immune System
Chairs: Markus Radsak (Mainz, Germany), Mustafa Diken (Mainz, Germany)
12:15 - 12:30
Sibel Mete (#216) University of Zurich (Zurich, Switzerland) Zoledronate nanoparticles repolarize neutrophils in tumor microenvironment
to impair growth of tumors
12:30-12:45
Ivan Shevchenko (#184) German Cancer Research Center and University Hospital
Mannheim (Heidelberg, Germany) Extracellular adenosine metabolism mediated by myeloid-derived suppressor cells
in melanoma and pancreatic cancer
12:45-13.00
Thomas Effert (#185) Johannes Gutenberg University (Mainz, Germany) Development of resistance towards Artesunate in MDAMB-231 human breast cancer
cells treatment
13.00-13:15
Cristina Maccalli (#205) San Raffaele Foundation Scientific Institute (Milan, Italy) Immunomodulatory properties of cancer stem cells isolated from human glioblastoma
and colorectal cancer
13:15-13:30
Anna I. Hooijkaas (#207) Netherlands Cancer Institute (Amsterdam,Netherlands) Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in
a mouse model of human melanoma
12:15-13:30
28
Hall C
(organized by Becton Dickinson)
Hinrich Abken University of Cologne (Cologne, Germany) CARs with extraordinary performance: T-cells redirected for an anti-tumor attack
15:00-16:30
Hall A
Holger Hoff TRON (Mainz, Germany)
Targeting inhibitory phosphatases by ectopic expression of miR181a augments
T-cell functionality
Plenary Session 7 / Immunoguiding
Chairs: Christian Ottensmeier (Southampton, USA),
Sjoerd van der Burg (Leiden, Netherlands)
15:00-15:30
Scott Tanner University of Toronto (Toronto, Canada)
An introduction to mass cytometry: fundamentals and applications
15:30-16:00
Robert Vonderheide University of Pennsylvania School of Medicine (Philadelphia, USA)
Cancer inflammation and immunosurveillance in pancreatic carcinoma in mice
and humans
16:00-16:30
Michael Kalos University of Pennsylvania School of Medicine (Philadelphia, USA)
Integrated biomarker analyses of T-cell therapy trials
16:30-17:00
Hall A
Closing Words
Christoph Huber (CIMT Chairman, Mainz)
29
CIMT | Floor Plan
CIMT | Program Overview
Floor Plan
Program Overview
D
08:30 - 09:00
G
A
C
B
E
Day 1 - May 23
Day 2 - May 24
Day 3 - May 25
Welcome Coffee and Addresses
Session 3: Antibodies
Session 5:
Improving Immunity
M. Glennie, T. Raum, M. Sliwkowsky
09:00 - 09:30
09:30 - 10:00
S. Akira, S. van der Burg, H. Schild,
R. Offringa
Opening lecture: P. Srivastava
10:00 - 10:30
Session 1: Tumor Vaccination
Coffee Break
10:30 - 11:00
T. Keler, J. Berzovsky, J. Allison,
R. Schreiber
Short Talk Sessions I-II-III
Short Talk
Sessions
IV-V-VI-VII
11:00 - 11:30
11:30 - 12:00
H
F
12:00 - 12:30
Breakout “CIP”
12:30 - 13:00
J. Galon, C. Gouttefangeas,
S. Janetzki, S. Hadrup, N. Bidmon, C. Ottensmeier, S.Walter
Lunch
Break
14:00 - 14:30
Industry Satellite
Symposium I
I. Melero, R. Stripecke,
T. Feuchtinger
Session 2:
Individualized Medicine
K. Pantel, L. Zitvogel, K. Itoh
Session 4: Cellular Therapies
C. June, P. Hwu, M. Nishimura, W. Herr
14:30 - 15:00
Label:
Location:
15:00 - 15:30
Poster Session I
15:30 - 16:00
Hall A
B
Hall B
C
Hall C
D
West Foyer
17:30 - 18:00
E
South Foyer
18:00 - 18:30
F
Registration
G
H
North Foyer
Foyer
16:30 - 17:00
Industry
Satellite
Symposium II
Lunch Break
by Becton Dickinson,
H. Abken, H. Hoff
Session 7: Immunoguiding
Coffee &
Break
S. Tanner, R. Vonderheide, M. Kalos
Poster Session II
16:00 - 16:30
A
Session 6:
Regulatory
Research
P. Bross, S. Khleif,
U. Sahin, U. Kalinke, C. Britten,
B. Flamion
Lunch
Break
by Miltenyi Biotec
13:00 - 13:30
13:30 - 14:00
Coffee Break
Feature Lecture
Coffee
Break
Closing Words
T. Gajewski (until 17:10)
17:00 - 17:30
Late Session
Immunomics
R. Kralovics, R. Scheuermann,
Z. Trajanoski, J. Castle
(start 17:15 - 19.15)
Keynote Lecture: A. Lanzavecchia
18:30 - 19:00
19:00 - 19:30
19:30 - 20:00
Social Event
20:00 - 24:00
30
31
CIMT | Sponsors, Partners & Supporters
CIMT | Industry Exhibitors
Sponsors, Partners & Supporters
We gratefully acknowledge the recurring support from our sponsors and partners.
Partners
Industry Exhibitors
Sponsors
Forschungszentrum
Immunologie Mainz
Supporters of CIP
32
33
Poster Presentations:
Abstracts have been selected for poster presentation
in six categories:
Cellular Therapy
Improving Immunity
Immunomonitoring
New Targets & New Leads
Therapeutic Vaccination
2012 CIMT
Poster Award
Tumor Biology & Interaction with the Immune System
sponsored by
Authors are required to be present at both poster sessions
May
23, 3 pm - 4:30 pm and May 24, 4 pm - 5:30 pm
34
Forschungszentrum
Immunologie Mainz
CIMT | Abstract List
CIMT | Abstract List
Abstract List (001 - 023)
Cellular Therapy
No.:
Short talk:
Abstract list (024 - 043)
Cellular Therapy
Title:
No.:
001-New regulatory compliant technology for isolation of cells in Cell Therapy using Dynabeads
002yesSuccessful Treatment of Melanoma Brain Metastases with Adoptive Cell Therapy
003yesγ9- and δ2-CDR3 domains regulate functional avidity of T-cells harbouring γ9δ2T-cell receptors
004-Generation of retroviral vectors encoding WT1-specific TCRs for the transduction of mature
T-cells
Short talk:
Title:
024-In vitro activation of NK cells overcomes resistance of multicellular Ewing sarcoma sphere
architecture to NK cell lysis
025-Animal-component free GMP-grade medium for the activation and expansion of T-cells
026-Immune-dominant Wilms’ Tumor 1 (WT1) peptide as target structure for cellular immune
therapy in acute myeloid leukemia (AML)
027-Establishment of an efficient transient transfection method for preparation of therapeutics
expressing T lymphocytes
005-Clinical grade production of human mesenchymal stem cells
006yesRetargeted Natural Killer Cells for Adoptive Immunotherapy of Glioblastoma
028-In vivo sunitinib pretreatment improves expansion of tumor infiltrating lymphocyte from
renal cell carcinoma patients
007-T-cell Activation and Expansion by use of Dynabeads®CD3/CD28 CTS™ for
Immunotherapy applications
029-Immunosuppressive effects of antifungal agents on murine CD8 T-cells in vitro and in vivo
008-Melanoma-specific bone marrow memory T-cells exert antitumor activity in ret transgenic mice
030-A novel process technology for automated NK cell culture and enrichment
+
+
+
009-Induction of polyfunctional CD4 and CD8 T-cell responses against NY-ESO-1 for adoptive
T-cell transfer in cancer patients
031-A group of T-cell receptors with strict requirements in the hypervariable region, but plasticity
in the germline encoded domains, for recognition of HLA-A2/CD20
010-A dual role for rolipram in modulating T-cell responses in murine Graft-versus-Host Disease
032-Analysis and optimization of a p53264-272- tumor antigen-specific single chain TCR in terms
of TCR mispairing in human T-cells
+
011-Everolimus inhibits CMV specific CD8 T-cells
033-Effect of cord blood regulatory T-cells on natural killer cell differentiation and function
+
012-Polyoma-Virus-specific CD8 T-cells for Cellular Immunotherapy
+
013yesTargeting leukemia using allo-HLA-DQ and allo-HLA-DP specific CD4 T-cells
034-An optimized single chain p53(264-272)-specific T-cell receptor devoid of ON/OFF target
autoimmunity in a humanized mouse model of adoptive T-cell transfer
+
014-HLA-DP antigens are major targets of AML-reactive CD4 T-cells isolated from HLA-DR/DQ
matched donors in vitro
035-Identification of donor-derived antigen-specific CTLs for clinical application using cysteine
modified tetramers
+
015-Human AML-reactive CD8 cytotoxic T lymphocyte clones effectively reduce leukemic burden
in NSG mice
016-Adoptive lymph node-derived HPV-specific T-cell therapy for cervical cancer
036-Reprogramming T-cells with an optimized Melanoma-specific human single chain T-cell
receptor results in substantial tumor cell recognition but also in mispairing with endogenous
TCR chains
017-Depletion of naive T-cells using GMP grade CD45RA MicroBeads
037-Targeting dendritic cells with functionalized nanoparticles
018-NK cell subpopulations differ in their tissue specific homing capacity and impact
in GVHD prevention
038-CD8 T-cell-specific transfer of TCR genes enhances tumor cell killing
019-rhIL-15 and rhIL-2 Enhance the Growth and Function of TCR Transduced T-cells
for Adoptive Immunotherapy
020-Reduced alloreactivity of human memory versus naive CD8 T-cells in vitro as well as in vivo:
Defining optimal target-populations for TCR-transfer
021yesDevelopment of clinically implementable imaging strategies for T-cell receptor-transgenic T-cells
022yesAdoptive transfer of redirected T-cells targeting a TGFβRII frameshift mutation frequently
occuring in microsatellite instable colon cancer
+
023-Donor lymphocyte infusion induces polyspecific CD8 T-cell responses with concurrent
molecular remission in AML with NPM1 mutation
+
039yesNext Generation Sequencing of γδ T-cell receptor repertoires
040-In vitro “on-target”-reactivity of affinity-modified p53264-272 tumor antigen-specific TCRs
retrovirally transduced into human T-cells
041-Cellular and molecular events controlling acquisition of cytotoxic activity by tumour-reactive
+
CD4 T-cells during melanoma progression and immunotherapy
042yesGeneration of MCSP-specific, MHC-independent T-cells by RNA electroporation at a clinically
feasible scale
+
043-Reprogramming bulk CD8 and g/d T lymphocytes with a specificity for adenovirus by
electroporation of TCR-encoding mRNA
*
personalized medicine-short talk extra category
36
37
CIMT | Abstract List
CIMT | Abstract List
Abstract List (044 - 064)
Enhancing Immunity
No.:
Short talk:
Abstract List (065 - 086)
Enhancing Immunity
Title:
No.:
Short talk:
Title:
044yesImmunotherapeutic potential of fully human anti-Macrophage Migration Inhibitory Factor
antibodies in different mouse cancer models
065-The Chemotherapeutic Compound 2-Deoxy D-Glucose Prevents Cell Surface Expression of
NKG2D Ligands through Inhibition of N-Linked Glycosylation
045-Antibody fusion proteins of cytokines and costimulatory ligands for cancer immunotherapy
066-Direct immunomodulatory effects of zoledronic acid on NK cells in Ewing sarcoma
046-Single-chain bispecific antibodies activate human regulatory T-cells and trigger their
suppressive function
067-Enhancement of cellular immune response against tumor cells using PAMPs
047-Immunomodulation of peripheral blood mononuclear cells by PHA and irradiated K562
048-Heterologous adeno-poxvirus combination for immunogenic cancer targeting
068-Optimized human Granzyme B based immunotoxin to circumvent the inhibitory potential of
SerpinB9 during cancer therapy
069-CTLA-4 antibodies Tremelimumab and Ipilimumab overcome tumor-mediated immunsuppres
sive effects on human dendritic cells
049-Immunotherapeutic synergy between anti-CD137 mAb and intratumoral admistration of a
cytopathic Semliki Forest virus encoding IL-12
070-Targeting of Macrophage Galactose-type C-type Lectin (MGL) induces DC signaling and activation
050-Antitumoral responses against liver implanted tumors induced by Semliki Forest virus
expressing IL-12 can be potentiated by coadministration of IL-15
071-Exploring the feasibility of future combinatorial approaches of chemotherapy with immuno
therapy for melanoma – influence of chemotherapeutic drugs on the functions of immune cells
+
051-Tumor-specific CD4 T-cells develop cytotoxic activity and eliminate virus-induced tumor cells
in the absence of regulatory T-cells
072-Updated results from a phase 2-3 clinical study in patients with advanced colorectal carcincoma
treated with the immunomodulator MGN1703 – the IMPACT study
052yesCpG-ODN induced upregulation of BTLA mediating selective inhibition of human B cells
073yesEffective chemo/immunotherapy in melanoma patients activates non-canonical AKT pathway
+
of Melan-A+ tumor-specific CD8 T-cell clones
053-Constitutive activation of the NFkB pathways in DC improves their T-cell stimulatory capacity
and IL-12p70 secretion
+
+
054-A concomitant interaction of CD4 T-helper cells, DC, and CD8 T-cells is required for an
+
effective boosting of tumor antigen-specific CD8 T-cell expansion
055-Non-toxic application of paclitaxel reduces chronic inflammation and abrogates myeloid
derived suppressor cell activity in ret transgenic melanoma bearing mice
056-CD86 and IL-12p70 are key players for T helper 1 polarization and NK cell activation
by TLR-matured dendritic cells
057-Prestimulation with 2-Hydroxy-Octadecylphosphocholine inhibits the Cytokine Secretion of
Monocyte-derived Dendritic Cells matured with Dendrophilin A201 and IFN-γ without
Changing their TH1-Polarisation
058-Paclitaxel potentiates endotoxin-induced maturation of human monocyte-derived dendritic
cells under serum-free condition
059-The bacterial preparation OK432 induces IL-12p70 secretion in human dendritic cells in a TLR3
dependent manner
060yesZoledronic acid-treated monocytes augment TRAIL-mediated cytotoxicity of human NK cells
061-Activation of the human immune system via Toll-like receptors by the oncolytic parvovirus
H-1 and its combination with targeted agents
+
062-Virus-specific CD8 T-cells up-regulate PD-1 expression during acute Friend retrovirus
infection but are highly cytotoxic and control virus replication
063yesSynergistic augmentation of CD40-mediated activation of antigen-presenting cells
by amphiphilic poly(γ-glutamic acid) nanoparticles
+
064-Inhibition of tumor immunity by tumor-infiltrating CD4 regulatory T-cells in patients with
primary and metastatic liver cancer can be abrogated by soluble GITR-ligand
38
074-New ways to improve the treatment of rectal cancer patients with liver metastases using
metronomic regimen of chemotherapy with recombinant inferferon-alpha
075-Combining Gemcitabine and RIG-I signaling for chemoimmunetherapy of pancreatic cancer
076-Immune-complexes formed by intracellular tumor antigens released by chemotherapy and an
injected antigen-specific antibody have an adjuvant effect to activate the adoptive immune system
against cancer
077-The Role Of Combining Synthetic Imidazoquinoline Toll-like Receptor (TLR) Agonists And
GM-CSF Activity For Potentiating Cell Activation In Autologous Cellular Immunotherapy (ACI)
078-Sharply discordant biological properties of synthetic noncoding dsRNA of different size:
translational opportunities in cancer
079yesmTOR inhibitor Rapamycin Enhances the Cancer Therapeutic Potency of Naked RNA Vaccine
080-CpG Oligodeoxynucleotides enhances both humoral and cellular immune responses against
FMDV in mice
081-Potent Stimulation of the Innate Immune System by Nucleic Acid Based TLR Ligands
Encapsulated in Nanoliposomes
082-CpG DNA containing nanoparticles as potential antitumor agents
083-Potentiating the immunostimulatory properties of CpG oligodeoxynucleotides: aiming to
develop a better vaccine adjuvant
084-A novel ODN delivery platform: CpG-ODN-loaded exosome nanovesicles
085-Bacteriocin DNA nanocomplexes as immunotherapeutic carriers
086-Alternative approaches to improve immunity against cancer and infectious diseases:
development of nucleic acid loaded nanocarrier systems
39
CIMT | Abstract List
CIMT | Abstract List
Abstract List (087 - 108)
Immunomonitoring
No.:
Short talk:
Title:
Abstract List (109 - 112)
Immunomonitoring
No.:
Short talk:
Title:
087-Divpenia (low TCR diversity) and lymphopenia as prognostic factors of OS in metastatic
breast cancer
109-Homing receptor expression by T-cells infiltrating the metastatic melanoma microenvironment
and relevance to combination immune therapy
088yesT-cell recognition of tumor-associated antigens in patients with preinvasive lesions of the breast
110-Detection of tumour antigen specific T-cell populations in leukaemia: markers of good prognosis?
089yesAntibodies against CTL epitopes from tumor-associated antigens were widely detectable in
humans: potential prognostic significance in cancer patients
111-TransHLA – A Method for determining the HLA genotype from RNA-Seq data
112-Predictive biomarkers for treatment success of the therapeutic renal cell cancer vaccine IMA901
090-Reliable assay for monitoring CMV-specific T-cell immunity following Allogeneic Hematopoietic
Cell Transplantation
091-Expression of Foxp3 in Colorectal Cancer but not in Treg Cells Correlates with Disease
Progression in Patients with Colorectal Cancer
*
personalized medicine-short talk extra category
092-Molecular signature of virus-specific T-cell polyfunctionality
093-Activation and frequency of Myeloid Subsets in peripheral blood is associated with clinical
outcome in prostate cancer patients treated with Prostate GVAX and ipilimumab
+
094-CD8 T-cells specific for peptides encoded by large T and small T antigen from Merkel cell
polyomavirus is detected in merkel cell carcinoma patients and not healthy individuals
095-No correlation between spontaneous tumor antigen-specific T-cell and antibody responses in
the majority of primary melanoma patients
096yesDissection of anti-CTLA4-induced cytotoxic T-cell responses in melanoma
097yesTracking T-cell clones using high-throughput sequencing of antigen receptorCDR3 chains
098-HLA subtype variation strongly affects MHC multimer-based monitoring of antigen
specific CD8 T-cell responses
099-Cancer vaccination with telomerase peptide GV1001: The immune response relates to clinical
outcome
100yesNY-ESO-1 and Melan-A-reactive T-cells are predictive for the clinical outcome of
late-stage melanoma patients
101-Reference samples to control performance of HLA-peptide multimer staining
experiments – First results from a proficiency panel testing
102-Reference Samples as stable controls for T-cell assay
103-Treg Flow Cytometry Staining – Hunting a FOX
104yesActivation, Dysfunction and Retention of Antigen-Reactive T-cells in the Vaccine Site Micro
environment after Multipeptide Vaccine in Incomplete Freund’s Adjuvant
105-Myeloid sub-populations are correlated to survival in patients with cervical cancer
106-Quantitative determination of tumor specific Treg in lymphatic compartments of patients
107yesIMA910, a novel multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple
+
+
CD8 and CD4 T-cell responses associated with improved survival
108-Immune Monitoring of Poxvirus based Cancer Immunotherapies
40
41
CIMT | Abstract List
CIMT | Abstract List
Abstract List (113 - 136)
New Targets and Leads
No.:
Short talk:
Abstract List (137 - 143)
New Targets and Leads
Title:
No.:
113-Regression of metastatic melanoma by targeting melanoma stem cells
114-EpCAM-targeting antibodies for the treatment of a murine model of spontaneous gastric cancer
115-Development of a novel modular cell targeting system for immunotherapy of acute myeloid
leukemia
+
116-HLA-restricted CD4 T-cell epitopes derived from cancer-retina antigen PDE6 alpha as
potential tools for T-cell based immunotherapy approaches
Short talk:
Title:
137-Frequently recognized MHC class II epitopes for an easy generation and detection of EBV
+
specific CD4 T-cells
138-High throughput in vitro priming of tumor specific T-cells
139yesIdentification of a tumour-associated autoantibody signature with high diagnostic value for
early detection of gastric cancer
140-Spontaneous humoral antibody responses against tumor-associated antigens in malignant
melanoma patients
117
yes Targeting mutated and overexpressed tumor antigens in cancer immunotherapy
118
yes Mutated BRAF and NRAS proteins as possible targets for the immunotherapy of melanoma
141-A peptide microarray platform with a new robust data analysis package
119-Natural HLA ligands provide novel T-cell epitopes for immunotherapy of ovarian carcinoma
142-Human chorionic gonadotropin exerts immunosuppressive effects in a mouse model of Graft
versus-Host disease
120-Identification of new tumor specific HLA-Ligands – Separating tumor and stroma origin
143-Expression and impact of interleukin-22 in human lung cancer
121-HPV16 E6 and E7 T-cell epitope identification by mass spectrometry
122-Identification of NPM-ALK-reactive T-cells in children with NPM-ALK-positive-anaplastic large
cell lymphoma (ALCL)
*
personalized medicine-short talk extra category
123-Attenuation of Lethal Semliki Forest Virus Neurovirulence in Mice by Neuronal microRNA
Targeting
124-Siglec-7 and -9 on Natural killer cells and their ligands on tumor cells are novel inhibitory
regulators of human NK cell functions in vitro and of tumor cell killing in vivo
125-MELOE-1 contains multiple HLA class II T-cell epitopes eliciting Th1 responses in melanoma
patients
126-Identification of hematopoietic minor H antigens using leukemia reactive T-cells and genetic
linkage analysis
127-Identification of unique colorectal cancer T-cell antigens by next generation sequencing of
somatically mutated genes
128-Identification of lung cancer associated oncoantigens as targets for active immunotherapy
129-Designer host defense peptides for treatment of colorectal carcinoma
+
130-Characterization of CD4 T-cell responses specific for novel HLA-DR-restricted epitopes
derived from the breast tumor antigen NY-BR-1
+
131yesEpitopes derived from the mutated region of Nucleophosmine 1 (NPM1) induce both CD4
+
and CD8 T-cell responses
+
132-CD4 T-cells Recognising Human B Lymphoma-Associated Antigens
133-Novel tumor associated antigens for chronic lymphocytic leukemia
134-The Quest for Novel Peptide Vaccines in Renal Cell Carcinoma: Mining the HLA‑Ligandome
135-The HLA class I ligandome of prostate cancer: New targets for peptide-based immunotherapy
136yesLymphoma specific immunity in healthy individuals and patients targets phosphorylated
antigens derived from the cytoplasmic tail of CD19
42
43
CIMT | Abstract List
CIMT | Abstract List
Abstract List (144 - 162)
Therapeutic Vaccination
No.:
Short talk:
Title:
Abstract List (163 - 179)
Therapeutic Vaccination
No.:
Short talk:
Title:
144-Tumor cells infected by Measles virus vaccine induce plasmacytoid dendritic cell (pDC)
maturation and tumor antigen cross-presentation
163-Immunological results of a phase 1-2 therapeutic vaccination study by MGN1601 in patients
with advanced renal cell carcinoma
145-Use of Oncolytic Rhabdoviruses as Potent Tumour Vaccine Boosters
164-Long Peptides Complexed with A Novel Delivery Sytem CHP Nanogel Leads to the Improved
Vaccine-induced Specific Immune Responses with CpG oligo DNA or poly-I:C RNA
146-HLA-A*0201+ plasmacytoid dendritic cells provide a cell-based immunotherapy for melanoma
patients
147-Intravaginal immunostimulation after vaccination increase local vaccine-specific CD8 T-cells
and tumor regression of genital tumors in mice
148-Characterization of the human CD83 promoter complex for transcriptional targeting of dendritic
cells in vivo
149-Stimulation of dendritic cells with vaccine and vaccine-antibody complex and effect on
immune response
150-A pilot study of peptide-based vaccines in combination with poly ICLC in patients with WHO
grade 2 low-grade glioma
151yesActively personalized multi-peptide vaccination for primary liver cancers – a novel strategy for
overcoming residual disease
152-Induction of antigen-specific T and B cell responses in mice with a reconstituted human
immune system
153-Vaccines for tumour prevention: Does Indoleamine 2,3-Dioxygenase (IDO) silencing enhance
DNA vaccination?
154-DCONE: Next generation off-the-shelf dendritic cell vaccine applicable for a broad range of
cancer indications
155-Expansion of polyfunctional antigen-specific T-cells upon stimulation with mRNA
electroporated dendritic cells in the presence of immunomodulatory drugs
156yesTargeting Cancer Stem Cells by raising cytotoxic T-cell responses against the stemness protein SOX2
157-Comprehensive preclinical model evaluating a protein-based PRAME specific cancer immuno
therapy to fight against PRAME expressing tumors
158-Combining common chemotherapeutic regimens with immunotherapy – assessment of the
immunological effects of FOLFOX, FOLFIRI and cisplatin
159-RNAdjuvant®: a novel, highly-potent, RNA-based adjuvant supports induction of balanced
immune response (TH1 and TH 2) and anti-tumor activity
165-Preliminary results of a triple peptide escalating dose vaccination Phase I/II clinical trial as
consolidation treatment in women affected by ovarian cancer
166-Immune responses against frameshift antigens in microsatellite unstable colorectal cancers
167-Sipuleucel-T product characterization across different disease states of prostate cancer
168-Construction of DNA vaccines expressing the novel tumor antigen neuroplastin: protective
efficacy against mammary adenocarcinoma in immunized mice
169-Dendritic-tumor cell hybrids in therapeutic vaccination against advanced neuroblastoma
170yesActive Immunotherapy of Cancer with PROSTVAC® and MVA-BN®-HER2 Demonstrate Potent
Preclinical Anti-tumor Efficacy
171-Development of a DC-based therapeutic vaccine for AML patients: Charakterization of
GMP-grade TLR-agonist matured 3-day DCs expressing the leukemia-associated antigens WT1
and PRAME
172-Dendritic cell vaccine after induction chemotherapy in patient with metastatic melanoma:
prospective randomized single-institution trial
173yesA Novel Minigene Scaffold for Cancer Vaccine Applications
174-Generation of immunogenic MUC1 glycopeptides by DCs primed with microvesicle bound
MUC1 tumor associated glycoprotein, but not with the soluble MUC1
175yesTumor immunity conferred by mRNA-transfected dendritic cells expressing bi-functional
polypeptides which couple MHC-I presentation to dendritic cell activation
176-A First In Man Phase I Trial Of IMA950 (A Novel Multi-Peptide Vaccine) Plus GM-CSF In
Patients With Newly Diagnosed Glioblastoma – Design And Preliminary Results of a Cancer
Research UK Study
177-Induction of anti tumor responses against malignant melanoma via antigen targeting in vivo
178-Comparison of clinical grade polarized and standard matured dendritic cells for cancer
immunotherapy
179-Investigating the functionality of tumour-infiltrating lymphocytes induced by immunotherapy
160-Dendritic cell vaccination in melanoma patients: mRNA electroporated dendritic cells
improves immunological and clinical responses
161-IVAC – Individualized Vaccines for Cancer
162yesIntradermal immunization with a novel mRNA-based vaccination technology induces strong
T- and B-cell responses in phase I/IIa trials in non-small-cell lung cancer (NSCLC) and
prostate carcinoma (PCA)
44
45
CIMT | Abstract List
CIMT | Abstract List
Abstract List (200 - 216)
Tumor Biology and Interaction
with the Immune System
Abstract List (180 - 199)
Tumor Biology and Interaction
with the Immune System
No.:
Short talk:
Title:
No.:
Short talk:
Title:
180-Study of the involvement of the activity of oxygen free radicals in the development of colorectal
cancer: chemo-preventive effect of an antioxidant SOD mimetic a Glisodin
200-Helicobacter-induced preneoplastic gastric immunopathology is suppressed by TLR2-activated
B cell induced T regulatory-1 cells
181-The effects of ADAM10 and Neprilysin on tumor-induced release of IL-6 and IL-10
201-Investigation and inhibition of tumor immune escape from NKG2D-dependent NK cell cytotoxicity
182-The Immunomodulatory Role of Endogenous Glucocorticoids in Ovarian Cancer
202-Impaired expression of TAP-2 as posttranscriptionally controlled by microRNAs, modulate
immune escape mechanisms in esophageal adenocarcinoma
183-Characterization of breast cancer stem cells and their correlation with circulating tumor cells
203-Expression and regulation of arginine transport proteins in human T lymphocytes
184yesExtracellular adenosine metabolism mediated by myeloid derived suppressor cells
in melanoma and pancreatic cancer
204-Arginine auxotrophy: tumor growth analysis in a 3D in vitro culture system
185yesDevelopment of Resistance towards Artesunate in MDAMB-231 Human Breast Cancer Cells
Treatment
205yesImmunomodulatory properties of cancer stem cells isolated from human glioblastoma and
colorectal cancer
186-Characterization of dormant melanoma cells and their interaction with memory CD8 T-cells in
ret transgenic mouse melanoma model
206-Radiation-induced gene expression leads to altered immune signaling and migration in
glioma-initiating cells
187-Cyclophosphamide-induced myeloid-derived suppressor cells: their functional characterization
and modulation by 5-azacytidine and IL-12
207yesSelective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model
of human melanoma
188-TLR3-expressing Tumor Parenchyma and Infiltrating NK Cells Promote Tumor Control in
Hepatocellular Carcinoma Patients
208-Synchronous BRAF V600E and MEK inhibition leads to superior control of melanoma by limiting
MEK inhibitor induced skin toxicity
189-A novel mitochondria-targeted antioxidant SkQ1: immunoregulatory properties in pancreatic cancer
209-Human skin-derived and lymph node-resident dendritic cell subsets display differential T-cell
stimulatory activity and are differentially modulated by primary melanoma tumors and metas
tases in the sentinel lymph node
190-B7-H1 in chemo/immune therapy of pancreatic cancer
191-Effect of platinum-containing chemotherapy on tumor micro-environment in gynecological
malignancies
210-Clinical implications of immune evasion in microsatellite-unstable colorectal cancer
192-HLA class I and II antigen expression in human bladder cancer
211-Cytokines in bone marrow and peripheral blood of breast cancer patients as the prognostic
signs of tumor progression
193-Epigenetic mechanisms underlying IFNγ-induced upregulation of antigen presenting machinery
genes in tumor cells
212-Investigation the interferon-alpha as a modifier of epithelial – mesenchymal transition of
malignant cells in vitro
194-Hyperthermic intraperitoneal chemotherapy in patients with peritoneal carcinomatosis:
Role of heat shock proteins and dissecting effects of hyperthermia
213-Dynamic Capture of Tumor Cell Propagation with Associated Stromal and Immune Responses
in CNS Tumor Microenvironment
195-Clinical significance and therapeutic potential of programmed death 1 and programmed death
ligand 1 and 2 expression in human colorectal cancer
214-Different tumor suppressors control expression of ULBP2, an innate stress ligand of the ­
activating lymphocyte receptor NKG2D
196yesCorrelation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with clinical
and pathological relationships of patients with oral squamous cell carcinoma
215-Regulation of natural killer cell development and antitumor immunity by the interleukin 15
system
197-It takes two to tango: MHC-I and Invariant chain in harmony
216yesZoledronate Nanoparticles Repolarize Neutrophils in Tumor Microenvironment to Impair
Growth of Tumors
198-Endothelial cells derived from non-malignant tissues as models for tumor vasculature are of
limited value
199-Cytotoxic dendritic cells inhibit regulatory T lymphocyte generation
46
*
personalized medicine-short talk extra category
47
001 | Cellular Therapy
New regulatory compliant technology for isolation of cells in
Cell Therapy using Dynabeads®
Cellular Therapy
Karoline Schjetne, Lars Norderhaug, Grethe Økern, Kerstin Bernstrom, Eli Lien, Berit Grøn,
Mark Bonehaydi, Tanja Aarvak
Life Technologies, Oslo, Norway
Introduction: Cell therapy is experiencing a
Conclusion: By using a highly specific detach
state of resurgence, driven by a series of positive
buffer, both magnetic beads and antibodies can
achievements as recently demonstrated in Chronic
be removed from the cells after isolation leaving
Lymphatic Leukemia (CLL) patients treated with
a highly pure cell product. This novel technology
autologous redirected T-cells (University of Penn-
will represent a regulatory compliant technology
sylvania). Purification of cells before administra-
that can be implemented into a GMP manufactur-
tion into patients is of paramount importance to
ing processes for a variety of differenT-cell drugs.
ensure efficiency and safety of the treatment. For
Dynabeads can be customized for many applica-
stem cell therapy, tumorigenicity can be avoided if
tions by conjugation of antibodies specific for the
the pluripotent undifferentiated cells are removed
targeT-cell of your choice.
from the stem cell-derived cell drug. To assure
pure and safe cell drugs there is a growing need to
develop a GMP-complianT-cell isolation technology
which enables clinicians to produce cell drugs free
of antibodies and magnetic beads prior to infusion
into the patients.
+
Results: CD3 T-cells were isolated by Dynabeads
conjugated with a recombinant antibody specific
for human CD3. The T-cells were captured by the
Dynabeads on a DynaMag™ magnet and unbound
CD3
neg
cells were removed by washings. Dynabeads
were removed from the cells by adding a specific
detach buffer to enable a bead- and antibody-free
+
cell product. The isolated CD3 T-cells were stimulated by Dynabeads CD3/CD28 beads and proliferation was analyzed. Recovery of targeT-cells was
65% (range 36-87) and purity >98% (range 96-99).
51
002 | Cellular Therapy
003 | Cellular Therapy
Successful Treatment of Melanoma Brain Metastases with
Adoptive Cell Therapy
γ9- and δ2-CDR3 domains regulate functional avidity of T-cells
harbouring γ9δ2T-cell receptors
1
1
1
1
1
1
1
1
1
1
Jenny J. Hong , Steven A. Rosenberg , Mark E. Dudley , James C. Yang , Donald E. White ,
2
1
John A. Butman , Richard M. Sherry Cordula Gründer , Suzanne van Dorp , Samantha Hol , Esther Drent , Kirsten Scholten ,
1
1, 2
3
1
Sabine Heijhuurs , Anton Martens , Roland Stong and Jürgen Kuball 1
National Cancer Institute, NIH, Surgery Branch, Bethesda, Maryland, USA
1
Department of Haematology and Immunology, UMC Utrecht, Utrecht, Netherlands
The Clinical Center of the NIH, Radiology and Imaging Sciences, Bethesda, Maryland, USA
2
Department of Cell Biology, UMC Utrecht, Utrecht, Netherlands
3
Department of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
2
52
Introduction and purpose: Metastatic melanoma to
Results: Seventeen of these 26 patients received ACT
Immunotherapy with innate immune cells has
the brain is a difficult and frequent clinical chal-
with TIL. Seven of these patients (41%) achieved a
recently evoked a broad interest as a novel treat-
within the CDR3 domains of a γ9δ2TCR were iden-
lenge. Current systemic treatments for patients
complete response in the brain, and six patients
ment option for cancer patients. γ9δ2T-cells are an
tified, suggesting a thus far underestimated role of
with melanoma and brain involvement are limited
achieved an overall partial response. In the nine
emerging innate cell population with strong anti-
δ2-CDR3 in particular in antigen-recognition. This
and largely ineffective. Adoptive cell therapy (ACT)
patients that received TCR-transduced lympho-
tumor reactivity, which makes them a promising
knowledge allowed improved tumor control by
with a nonmyeloablative preparative regimen
cytes, two patients achieved a complete response
candidate for immune interventions. γ9δ2T-cell re-
using engineered T-cells, not only in vitro, but also
using activated lymphocytes and interleukin-2 is
in the brain (22%) and one of these two achieved
ceptors (TCR) in particular recognize a broad panel
in vivo in a humanized mouse model.
an experimental approach that has been shown to
an overall partial response. One patient developed
of tumor cells with high avidity, and are therefore
be effective for some patients with metastatic mela-
a
hemorrhage
clinically attractive, since αβT-cells can be effi-
noma. The purpose of this study is to determine
during the thrombocytopenic phase of therapy and
ciently redirected against a variety of tumor cells
the objective response rate and response duration
had an uneventful metastatectomy.
by introducing a γ9δ2TCR. Here we demonstrate
of melanoma brain metastases to adoptive cell
Conclusion: ACT with a nonmyeloablative prepara-
that distinct γ9δ2TCRs mediate different functional
therapy (ACT) with autologous antitumor lympho-
tive regimen using either TIL- or TCR gene–trans-
avidities, and present the concept of combinatorial-
cytes plus interleukin-2 following a lymphodeplet-
duced cells and interleukin-2 can mediate complete
γδTCR-chain-exchange
ing preparative regimen.
and durable regression of melanoma brain metas-
method to create γ9δ2TCRs that mediate strong an-
Methods: Between 2000 and 2009, 264 patients
tases. This strategy can be used safely in selected
ti-tumor-responses. In this way, γ9- and δ2-chains
with metastatic melanoma received ACT, consist-
patients with metastatic melanoma to the brain.
derived from individual γ9δ2T-cell clones are newly
tumor-associated
subarachnoid
(CTE)
as
an
structurally and functionally important residues
efficient
ing of cyclophosphamide and fludarabine with or
combined, allowing the design of g9d2TCRs that
without total body irradiation, followed by the infu-
mediate a higher functional avidity against a broad
sion of autologous tumor-infiltrating lymphocytes
tumor cell panel in vitro and in vivo when com-
(TIL) or autologous peripheral blood lymphocytes
pared to a reference γ9δ2TCR. In addition, we dem-
retrovirally transduced to express a T-cell receptor
onstrate that this phenomenon is selectively caused
(TCR) that recognized the melanocyte differentia-
by differences in the CDR3 domains of g9- and d2-
tion antigens gp-100 or MART-1. From this group,
chain. Accordingly, alanine-scanning-mutations
26 patients were retrospectively identified to have
were performed to elucidate important residues
had untreated brain metastases and extracranial
within the CDR3 sequence and the impact of the
disease before receiving ACT. The response rate
CDR3 length for optimal g9d2TCR function. While
and duration of melanoma brain metastases, as
length and sequence seem to both play critical roles
well as the overall response rate, response dura-
in d2-CDR3, selectively the g9-CDR3 sequence but
tion, and survival for these patients, are presented
not the length is a crucial factor. To summarize,
53
004 | Cellular Therapy
005 | Cellular Therapy
Generation of retroviral vectors encoding WT1-specific TCRs for
the transduction of mature T-cells
Clinical grade production of human mesenchymal stem cells
1
2
3
1
3
Martina Anzaghe , Sebastian Newrzela , Björn-Philipp Kloke , Ben Eising , Winfried Wels 1
and Peter Bader Somchai Sangkitporn, Siripakorn Sangkitporn, Acharaporn Dumbua, Areerat Sangnoi,
­Sawitree Duangruang, Supaluk Buasrikaew, Apichat Chotchusri,
1
Goethe University, Hospital for Children and Adolescents III, Division of Stem Cell Transplan­
tation Frankfurt, Germany
Clinical Research Center, Department of Medical Sciences, Nonthaburi, Thailand
2
Goethe University, Senckenbergisches Institut für Pathologie, Frankfurt, Germany
3
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany
54
WT1 mRNA is highly expressed in over 96 human
TCR, the β-chain is combined with its matching
Stem cell-based therapies hold tremendous promise
ination. Endotoxin level did not over the critical
leukemias, including acute myelogenous leuke-
α-chain, connected via a P2A-peptide sequence for
for the treatment of human diseases. Mesenchymal
limit. Resistance to spontaneous transformation
mia (AML), acute lymphocytic leukemia (ALL), or
bicistronical expression in the gamma-retroviral
stem cells (MSCs) are promising candidate cells for
into tumor cells was monitored by karyotyping
chronic myelogenous leukemia (CML). Thus, WT1
vector MP91mcs-wPRE. We further modified MP91-
anticancer agents. They can exhibit a tropism to
and maintenance of normal morphology/ pheno-
was identified as a prognostic factor and a marker
wPRE and inserted a GFP cassette downstream to
cancer tissue and may serve as a cellular delivery
type after prolongs in-vitro culture.
for diagnosis of minimal residual disease (MRD) in
a internal ribosomal entrya site (IRES) resulting in
vehicle of various clinically relevant anticancer
In conclusion, the manufacturing process of
human leukemia. Immunogenicity of WT1 protein
modified MP91mcs-IRES-GFP-wPRE. TCR-encod-
factors. In cellular therapy, MSCs should be pro-
clinical grade MSCs was established at the DMSc
was demonstrated by humoral and cellular immune
ing gamma-retroviral vectors are packaged into
duced according to good manufacturing practices
stem cell facility. These clinical grade stem cells
responses naturally elicited in cancer patients. The
replication-deficient retroviral particles in a three-
(GMP) and good tissue practices (GTP). The main
will become the important tools for physicians to
evidence that WT1 is preferentially expressed in a
plasmid system with the help of packaging cell line
goal of the present study was to establish culture
develop the new therapeutic strategies; however,
variety of leukemic cells, renal cell carcinoma, lung
293T (HEK). After particle production human T-cell
processes for human autologous MSCs expansion
the strict regulations during cell processing have
cancer, ovarian tumors and malignant mesothelio-
line PM1 was transduced and GFP, CD3 and TCR
for use in clinical studies. All parts of the processes
to be followed in order to guarantee the highest
ma, but not in most normal cells suggests that WT1
expression was monitored via flow cytometry.
must be defined: the starting bone marrow samples
quality and safety for patients.
might be an attractive target to develop efficacious
The demonstration that cloned TCR genes can
and other materials, donor validation, process con-
immunotherapy against leukemia or various types
be used to produce T lymphocyte populations of
trols during the development processes and the
of solid tumors. In a previous study we demonstrat-
desired specificity offers new opportunities for
batch release level. A minimum quality control
ed that WT1 peptide-specific T-cells can be generat-
antigen specific T-cell therapy and provides basic
included microbiological safety (sterility and my-
ed from healthy donor volunteers. These WT1 CTL
knowledge for design of lentiviral vector systems
coplasma), purity (endotoxin assay), identity (flow
showed antigen-specifity and cytotoxicity towards
such as LeGo vectors or GMP-adapted self-inacti-
cytometry), potency (MSC differentiation), total
tumor cell lines and peptide-loaded T2 cells.
vating (SIN) lentiviral vectors.
cell count and viability.
In this study we aim to design gamma-retroviral
After expansion with the classical media composi-
vector(s) encoding WT1-specific TCRs in order to
tion consists of a basal medium DMED and 10%
transduce mature T-cells. WT1 TCR-transduced
FBS for 21-30 days, FASC analysis results showed
T-cells will be analysed regarding specifity and
that the plastic adherent MSCs were negative for
avidity in vitro and in vivo to evaluate clinical feasi-
haematopoietic surface markers CD34 and CD45
bility of WT1 TCR-transduced CTL. Hence, we per-
and positive for the characteristic markers CD73,
formed sequence analysis of TCR-α and -β chains
CD90 and CD105. Expanded cells exhibited mul-
of CTL clones from two different WT1 peptide
tilineage differentiation into osteoblast, adipocyte
specifities (WT1p37 and WT1p126). We designed
and chondroblast. Cell numbers and cell viability
gamma-retroviral-based vectors encoding for WT1
were adequate for clinical use. Microbiological
p37 and WT1 p126 specific TCR-α/β chains. Per
assays demonstrated the absence of any contam55
006 | Cellular Therapy
007 | Cellular Therapy
Retargeted Natural Killer Cells for Adoptive Immunotherapy of
Glioblastoma
T-cell Activation and Expansion by use of Dynabeads®CD3/
CD28 CTS™ for Immunotherapy applications
1
1
2
1
1
Kurt Schönfeld , Congcong Zhang , Michael Burger , Sabrina Genßler , Christiane Sahm ,
1
3
4
5
4
Christian Brendel , Sonja Naundorf , Marcus Odendahl , Ulrike Köhl , Torsten Tonn ,
1
2
1
­Manuel Grez , Joachim P. Steinbach , Winfried S. Wels 1
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main,Germany
2
Institute for Neurooncology, University Hospital, Frankfurt am Main, Germany
3
EUFETS GmbH, Idar-Oberstein, Germany
4
Med. Faculty, Technische Universität Dresden and German Red Cross Blood Donation Service
East, Dresden, Germany
5
Pediatric Hematology and Oncology, University Hospital, Frankfurt am Main, Germany
56
Kerstin Bernstrom, Grethe Okern, Karoline Schjetne, Lars Norderhaug and Tanja Aarvak.
LifeTechnologies corp.
In addition to primary natural killer (NK) cells
diotherapy and chemotherapy with temozolomide.
Dynabeads® CD3⁄CD28 CTS™ are developed for ex
CAR T-cells has shown to have potent anti tumor
continuously growing cytotoxic cell lines such as
However, recurrence of GBM is very frequent,
vivo isolation, activation, and expansion of human
effects and can establish memory in patients with
NK-92 are being considered for adoptive cancer
and the median survival of glioblastoma patients
T-cells for use in various Immunotherapy appli-
advanced leukemia.
immunotherapy. High cytotoxicity of NK-92 has
is only 12 to 15 months. Therefore, more efficient
cations. The size of the Dynabeads® is similar
Clinical scale cGMP protocols using Dynabeads®
been shown against malignanT-cells of hema-
treatment modalities are urgently needed, with im-
to antigen presenting cells. This combined with
CD3⁄CD28 CTS™ are established utilizing bioreac-
tologic origin in preclinical studies, and general
munotherapy representing a promising alternative
conjugation of anti-CD3 and anti-CD28 antibodies
tor systems and DynaMag™CTS™ to remove Dy-
safety of infusion of NK-92 cells has been estab-
to standard therapy. Our ongoing work focuses on
enables Dynabeads® CD3⁄CD28 CTS™ to mimic an
nabeads® CD3⁄CD28 CTS™ after T-cell expansion
lished in phase I clinical trials. To enhance their
evaluating the efficacy and feasibility of systemic
in vivo antigen presenting cell that provides both
that enables the processing of T-cell products > 10
therapeutic utility, we previously generated ge-
and intratumoral application of NK-92/5.28.z cells
primary and secondary activation signal to the
L. Dynabeads® CD3⁄CD28 CTS™ is manufactured
netically modified NK-92 variants that express chi-
in ErbB2-positive glioblastoma xenograft models in
T-cells. The use of Dynabeads® CD3⁄CD28 CTS™
under GMP conditions and a Master File is held at
meric antigen receptors (CAR) specific for tumor-
vivo as a basis for further development of geneti-
has shown to have several advantages over activa-
the US Food and Drug Administration.
associated surface antigens such as ErbB2 (HER2),
cally modified NK cells as an adoptive cellular im-
tion methods with anti-CD3 antibodies where only
EpCAM, GD2 and CD20. Such CAR were composed
munotherapy for glioblastoma patients.
a primary activation signal is provided.
of a tumor-specific single chain antibody frag-
Dynabeads® CD3⁄CD28 CTS™ activates T-cells to
ment (scFv) fused via hinge and transmembrane
give 100-1000 fold expansion of ex vivo activated
domains to intracellular signaling proteins such
T-cells within 9-12 days without antigen presenting
as CD3 zeta chain or a composite CD28-CD3 zeta
cells. The expanded T-cells have properties compa-
fusion molecule. For development towards clinical
rable to in vivo activated T-cells and express recep-
applications, here we generated a lentiviral second
tors as CD28, CD27 and CD62L. As such, activation
generation CAR construct (5.28.z) with specificity
and expansion with Dynabeads® CD3⁄CD28 CTS™
for ErbB2, and established GMP-compliant proto-
enables the generation of T-cell products that are of
cols for transduction and expansion of NK-92 cells.
early memory phenotype associated with long term
An ErbB2-specific single cell clone (NK-92/5.28.z)
in vivo persistence, survival and effector functions.
was isolated, which showed high and selective cy-
For activation and expansion of T-cells to gener-
totoxicity towards ErbB2-expressing glioblastoma
ate gene modified T-cells, the use of Dynabeads®
cells and tumor cells of various other origins in
CD3⁄CD28 CTS™ enables higher gene transduction
vitro, as well as specific tumor homing in murine
efficiencies (~60%) as compared to anti-CD3 anti-
in vivo models. Glioblastoma multiforme (GBM)
bodies (~30%). Dynabeads® CD3⁄CD28 has been
is the most common and aggressive intracranial
implemented in several clinical gene transfection
malignant tumor in humans. Standard therapy for
processes for the generation of chimeric antigen
GBM includes maximal safe surgical resection, ra-
receptor (CAR) modified T-cells. The use of such
57
008 | Cellular Therapy
009 | Cellular Therapy
Melanoma-specific bone marrow memory T-cells exert
­anti­tumor activity in ret transgenic mice
Induction of polyfunctional CD4+ and CD8+ T-cell responses
against NY-ESO-1 for adoptive T-cell transfer in cancer patients
Anastasia Stemke, Barbara, Roider, Viktor Umansky
Simone Kayser , Cristina Boß , Michael Schumm , Vanya Icheva , Stefan Stevanović , Pe1
3
1
1
ter Lang , Hans-Georg Rammensee , Rupert Handgretinger , Tobias Feuchtinger 1
Skin Cancer Unit, German Cancer Research Center and University Hospital Mannheim,
­Heidelberg, Germany
1
1
3
1
University Children´s Hospital, of Tübingen, Germany
2
Department of Dermatology, University Hospital Tübingen, Germany
3
Department of Immunology, Institute for Cell Biology, University of Tübingen, Germany
Metastatic melanoma is a severe disease with a
has been demonstrated that PD-L1 expression
Adoptive T-cell therapy is a promising option to
restimulated with the tumor antigen and enriched
high rate of lethality. It is known to be resistant
is abundant on many murine and human tumor
treat cancer. In contrast to vaccinations, T-cells for
based on IFN-γ capture technique. Third, T-cells
to current therapies. Since melanoma is immu-
cells, myeloid derived suppressor cells and DC.
adoptive transfer are generated ex vivo to be re-in-
were expanded using autologous feeder cells in the
nogenic, the immunotherapy can be a promis-
We showed that mature DC generated from BM
fused into the recipient. This enables the treatment
presence of IL-2, IL-7 and Interleukin-15 (IL-15).
ing treatment possibility. Memory T-cells (MTC)
precursors expressed high amounts of PD-L1. To
of immunocompromized patients and the use of al-
T-cell specifity, function and proliferative capacity
have abilities to respond quicker to antigens and
avoid an impaired T-cell activation we incubated
logeneic T-cells to exploit graft versus tumor effects.
was analyzed by flow cytometry and cell superna-
to release a broader spectrum of cytokines than
DC with anti-PD-L1 antibodies for 24 h followed by
Transferring T-cells in lymphodepleted hosts post
tants were tested in multiplex cytokine analysis.
naïve T-cells. In this study, we used the ret trans-
the co-culture with T-cells. ELISPOT analysis and
stem cell transplantation furthermore offers access
The T-cell products showed high numbers of spe-
genic mouse melanoma model, which resembles
IFN-γ capture assay revealed a higher IFN-γ pro-
to homeostatic cytokines and eliminates regulatory
cifically IFN-γ and TNF-α
the pathological situation of human melanoma in
duction and therefore an increased T-cell activation
T-cells or myeloid suppressor cells. For sustained
and no FOXP3 expressing regulatory T-cells in
contrast to transplantation models. We have previ-
in vitro when PD-L1 was blocked. Furthermore, an
T-cell responses in vivo CD4 T helper (Th) cells are
flow cytometry. Multiplex cytokine analysis of cell
ously shown that the bone marrow (BM) is a major
increased level of CD69 expression was detected on
essential. Importantly the Th1 cytokines Interfer-
culture supernatants confirmed secretion of IFN-γ
site for the persistence of tumor-specific MTC in
T-cells 40 h after the co-culture with DC.
on-gamma (IFN-γ) and tumor necrosis factor-alpha
and TNF-α and furthermore demonstrated secre-
cancer patients. In addition, melanoma-specific
To determine the distribution of adoptively trans-
(TNF-α) do not only have effects in orchestrating
tion of other Th1 cytokines and chemokines like
MTC were also found in the BM of ret transgenic
ferred MTC in the ret transgenic melanoma mouse
cytotoxic T-cell responses but themselves mediate
IL-2, GM-CSF, CXCL10, CXCL8, and CCL2 but also
mice without macroscopic tumors. Therefore, we
model we labeled T-cells with the cell proliferation
anti tumor effects.
the Th2 cytokine IL-5. Both, CD4 and CD8 T-cells
isolated CD3 cells from the BM of ret transgen-
dye eFlour® 670 (CPD) and adoptively transferred
The aim of our study was the set up of a good
with an effector memory phenotype proliferated in
ic mice with and without visible tumors. In vitro
them into recipient mice. We were able to detect
manufacturing practice (GMP) conform protocol
response to NY-ESO-1. CD107a assays demonstrat-
generated BM-derived dendritic cells (DC) from
the presence of labeled cells in melanoma lesions
that allows the generation of polyclonal CD4 and
+
+
+
+
T-cells but no IL-10
+
+
+
+
ed cytotoxicity of Th1 cells. The T-cell products
C57BL/6 non-transgenic mice were pulsed with
by flow cytometry.
CD8 T-cells specific for tumor associated antigens
did not include alloreactive T-cells and specifity
tumor lysate or tyrosinase related protein (TRP)-
To investigate the antitumor activity of melanoma-
from healthy donors that can be applied to immu-
against NY-ESO-1 was confirmed by cross stimula-
2-derived peptide followed by the co-culture with
specific MTC in vivo we adoptively transferred DC-
nocompromized cancer patients in allogeneic set-
tion with NY-ESO-1 antigen from different sources.
freshly separated T-cells from transgenic mice. The
activated MTCinto tumor-bearing mice by an intra-
tings. Large scale generation of T-cells specific to
In summary GMP-conform generation of NY-ESO-1
MTC activation was analyzed after 40 h by IFN-γ
cardiac injection. Upon the adoptive transfer, mice
the tumor antigen NY-ESO-1 from healthy donors
specific T-cells was established and approved. Al-
secretion using ELISPOT and IFN-γ capture assay.
showed a significantly longer survival compared to
were performed according to current GMP regula-
though tumor associated antigens are potential self
The expression of the early activation marker CD69
the control group.
tions in a GMP facility.
antigens, it is possible to induce a functional Th1
was determined by FACS analysis.
We suggest that an adoptive transfer of melanoma-
The protocol is devided into three steps. It starts
response in T-cells from healthy donors. Adoptive
It is well known that melanoma microenvironment
specific MTC activated with antigen-loaded DC,
with a presensitization of peripheral blood derived
T-cell therapy against tumor associated antigens
is immunosuppressive. One of the suppressive
which were pre-treated with anti-PD-L1 antibodies,
T-cells with NY-ESO-1 in the presence of Interleu-
could have implications for multiple tumor entities
mechanisms could be represented by the interac-
can enhance an anti-tumor response and therapeu-
kin-2 (IL-2) and Interleukin-7 (IL-7) to get a detect-
in autologous as well as allogeneic treatment ap-
tion between programmed death ligand-1 (PD-L1)
tic efficacy in vivo.
able population of IFN-γ secreting T-cells in re-
proaches.
and its receptor programmed death-1 (PD-1). It
58
2
sponse to the tumor antigen. Second, T-cells were
59
010 | Cellular Therapy
011 | Cellular Therapy
A dual role for rolipram in modulating T-cell responses in
­murine Graft-versus-Host Disease
Everolimus inhibits CMV specific CD8+ T-cells
1
1
1
1
1
Michael Weber , Pamela Stein , Tobias Bopp , Edgar Schmitt , Hansjörg Schild ,
2
Markus P. Radsak 1
Institute for Immunology, Johannes Gutenberg University Medical Center, Mainz, Germany
2
IIIrd Dept. of Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany
1
2
1
1
1
Nan Jin , Romy Zurleit , Anita Schmitt , Georg Malcherek , Michael Schmitt 1
Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany.
2
Department of Hematology/Oncology, University of Rostock, Rostock, Germany.
Graft-versus-host disease (GvHD) is a key contribu-
Everolimus is a novel immunosuppressive drug
250 ng/ml inhibited the proliferation and IFNgam-
tor to the morbidity and mortality after allogenic
which is approved for solid organ transplantation
ma secretion of HLA-A2 restricted CMVpp65 spe-
hematopoetic stem cell transplantation (HSCT).
and recently used off-label for patients after hemat-
cific CD8 T-cells after one round of MLPC. The
Regulatory Foxp3 CD4 T-cells (Treg) suppress con-
opoietic stem cell transplantation (HSCT). Everoli-
inhibition was dose-dependent and complete in-
ventional T-cell activation and can control GvHD.
mus is also useful in reducing cyclosporine-related
hibition of the proliferation of CMV specific CD8
We analyzed Treg dependent mechanisms to attenu-
nephrotoxicity. As reactivation of cytomegalovirus
T-cells was observed within the therapeutic (nM)
ate acute GvHD: Irradiated BALB/c mice received
(CMV) plays a pivotal role for the outcome of pa-
range, comparable to prednisolone, a well studied
bone marrow and T-cells from C57BL/6 donors
tients after HSCT, we investigated in vitro to which
immunosuppressive agent. The frequencies of
and developed severe GvHD that was rescued by
extent everolimus affects CD8
+
+
the transfer of donor Treg cells or IL-10
-/-
Treg cells.
+
T-cell responses
+
+
+
CD8 CMVtetramer+ T-cells were equal to those
specific to CMV.
of CD137+CMVtetramer+ T-cells, also comparable
Further in vitro studies confirmed that Treg-mediated
Peripheral blood mononuclear cells (PBMCs) were
in numbers to CMV-specific IFNgamma-producing
suppression of alloresponses occurred independent
taken from the blood of HLA-A2/CMV seropositive
cells in ELISPOT assays.
of Treg or DC derived IL-10. Instead Treg communi-
healthy volunteers by Ficoll-Hypaque density gra-
Everolimus is a potent immunosuppressive drug
cated with DCs via gap junctions, resulting in func-
dient centrifugation and isolated using MACS® by
for the treatment of rejection or graft-versus-host
tional inactivation of DC by a metabolic pathway
anti-CD8 antibody labeled magnetic beads to get a
disease (GVHD) after HSCT. As a note of caution,
involving cyclic adenosine monophosphate (cAMP)
purified CD8 population. The CD8 cells were then
attention must be paid to early detection and pre-
that is modulated by the drug rolipram. Rolipram
stimulated with HLA-A*0201-restricted epitope from
emptive treatment of CMV reactivity in patients
suppressed allogenic T-cell activation indirectly
CMVpp65 (NLVPMVATV) in a mixed lymphocyte-
treated with everolimus as we demonstrate here
by enhancing Treg mediated suppression of DC
+
peptide culture (MLPC). After MLPC for 6 days, CD8
that everolimus strongly hampers CMVpp65 spe-
activation and directly by inhibiting responder
T-cells were re-stimulated overnight by CMVpp65
cific CD8 T-cells.
T-cell proliferation. Consequently, Treg dependent
peptides pulsed K562 cells stably transfected with
suppression of GvHD was greatly enhanced upon
HLA-A02 and analyzed by enzyme-linked immuno-
additional rolipram treatment. In conclusion, we
spot (ELISPOT) assays and multi-color fluorescence
propose that an important pathway of Treg mediat-
flow cytometry. ELISPOT assays were used to de-
ed control of GvHD is based on a contact dependent
termine the frequency of CMV-specific IFNgamma-
pathway modulated by cAMP. These data provide
producing cells. Cells were stained with anti-CD8
the basis for future concepts to manipulate allo-
and anti-CD137 antibodies, as well as CMVpp65 te-
genic T-cell responses to prevent GvHD.
tramer to detect activated CMV specific CD8 T-cells
+
+
+
+
by flow cytometry.
Adding everolimus at a concentration of 8, 50 or
250 nM corresponding to serum levels of 8, 50 or
60
61
012 | Cellular Therapy
013 | Cellular Therapy
Polyoma-Virus-specific CD8+ T-cells for Cellular Immunotherapy
Targeting leukemia using allo-HLA-DQ and allo-HLA-DP
­specific CD4+ T-cells
1
1
3
1
1
1
1
2
1
Jiju Mani , Georg Malcherek , Dominik Schneidawind , Anita Schmitt , Rosaely Casalegno2
2
1
Garduño , Michael Linnebacher , Michael Schmitt Andrea Bloetz , Eva Distler , Elke Schnürer , Felix Schier , Matthias Theobald ,
1
Wolfgang Herr 1
Universitätsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany
1
2
Universitätsklinikum Rostock, Medizinische Klinik III, Hämatologie und Onkologie, Rostock,
Germany.
3 Department of Medicine, University Medical Center of Johannes Gutenberg University,
Mainz, Germany
2
Department of Pediatric Surgery, University Medical Center of Johannes Gutenberg University,
Mainz, Germany
3
Universitätsklinikum Ulm, Klinik für Innere Medizin III, Ulm, Germany.
One of the major clinical complications in patients
new avenues for T-cell immunotherapies with BKV-
In allogeneic hematopoietic stem cell transplan-
of primary acute myeloid leukemia (AML) blasts,
after post hematopoietic stem cell transplantation
specific T-cells from healthy donors for patients
tation (allo-HSCT) patient and donor are usually
fibroblasts (FB) and keratinocytes (KC). IFN-γ se-
(HSCT) is the reactivation of viruses including
with HC or even PML. Moreover T-cell receptor
matched for the HLA class I molecules A/B/C as
cretion and cytotoxicity could only be observed for
CMV and polyoma viruses like BK virus (BKV), JC
cloning strategies and reprogramming of T-cells
well as for the HLA class II molecules DRB1 and
those targets that carried the HLA-DQ/-DP allele
virus (JCV) that may result in hemorrhagic cystitis
will enable us to circumvent the problem of donors
DQB1. In contrast, the HLA-DPB1 locus is still
used for T-cell priming, demonstrating the speci-
(HC) and lethal progressive multifocal leukenceph-
without detectable polyoma-virus specific T-cells
ignored in donor selection. Clinical studies have
ficity of this approach. While AML blasts were rec-
alopathy (PML), respectively.
responses.
demonstrated that disparities at HLA-DQB1 and
ognized independent of pre-incubating them with
+
T-cells response
distinct HLA-DPB1 alleles do not adversely affect
IFN-γ, recognition of FB and KC required IFN-γ
towards selected peptides using SIFPEITHI algo-
the outcome of allo-HSCT. It has also been shown
pre-treatment. The level of reactivity to AML blasts
rithm for HLA-A2 binding in a mixed lymphocyte
that HLA class II is predominantly expressed on
exceeded that to FB and KC, respectively. IFN-γ
peptide culture (MLPC). Preliminary results were
hematopoietic cells under non-inflammatory con-
secretion and cytotoxicity could be blocked by a
very promising. By fluorescence-activated cell
ditions. Thus CD4 donor T-cells recognizing pa-
CD4-specific monoclonal antibody. We further in-
sorting (FACS) we identified CD8 T-cells specific
tient-derived HLA-DQB1 or permissive HLA-DPB1
vestigated HLA class II expression on hematopoi-
for BKV peptide VP1p108- and JCV peptide VP1p100
mismatch alleles may primarily target leukemic
etic and non-hematopoietic cells by flow cytometry.
in one out of ten normal donors with a comparably
First, we evaluated the CD8
+
+
and hematopoietic cells, while sparing non-hemat-
HLA class II was not detected on primary FB, KC,
low frequency. CD8 T-cells were shown to prolif-
opoietic tissues. We used PBMC of healthy donors
and normal kidney cells (each n=10), but was ex-
erate, secrete IFNγ and kill targeT-cells in a cross-
to generate mature monocyte-derived dendritic
pressed at significant levels on primary AML blasts
reactive manner. These cells were successfully ex-
cells (DC), which underwent transfection with in
and B-cell lines (each n=10). Expression levels fol-
panded by cycles of restimulation.
vitro transcribed RNA coding for single HLA-DQ/-
lowed the hierarchy: DP>DR>DQ. Up-regulation of
It is our goal to broaden the approach and to com-
DP mismatch alleles by electroporation. These
HLA class II expression was observed on all cell
mence a comprehensive study using pools of over-
allo-HLA expressing DC were used to stimulate
types after pre-incubation with IFN-γ, but not after
+
+
lapping peptides derived from BKV and JCV capsid
autologous naive CD4 CD45RA+ T-cells in mixed
addition of TNF-α, IL-1β and IL-6. The approach
proteins like VP1, VP2 and VP3 and identify in-
lymphocyte reactions (MLR) in vitro (n=6). Rapidly
presented here appears suitable for generating al-
dividual T-cell responses in an HLA-independent
expanding MLR cells showed specific IFN-γ pro-
lo-HLA-DQ/-DP specific CD4 T-cell lines that rec-
strategy. T-cell specificities include specificities
duction upon recognition of allo-HLA-DQ/-DP mol-
ognize leukemia cells while presumably sparing
for immunodominant epitopes and ideally cover a
ecules as demonstrated by lack of immune reactiv-
non-hematopoietic cells under non-inflammatory
broad range of HLA-haplotypes.
ity to autologous DC expressing irrelevant allo-HLA
conditions. It may be of potential use in adoptive
+
62
rd
+
Virus-specific CD8 T-cells are expanded and en-
II molecules as well as by complete inhibition of
immunotherapy of allo-HSCT patients who express
riched by streptamer technology or, T-cells are
allo-DQ/-DP reactivity using HLA allele-specific
single HLA-DQ or permissive HLA-DP mismatch
reprogrammed with T-cell receptor specific for
antibodies. The allo-HLA-DQ/-DP specific T-cells
alleles.
polyoma virus BKV and JCV. These strategies open
were also analyzed for reactivity to a broad panel
63
014 | Cellular Therapy
015 | Cellular Therapy
HLA-DP antigens are major targets of AML-reactive CD4+ Tcells isolated from HLA-DR/DQ matched donors in vitro
Human AML-reactive CD8+ cytotoxic T lymphocyte clones effectively reduce leukemic burden in NSG mice
Yvonne Eichinger, Eva Distler, Elke Schnürer, Matthias Theobald, Wolfgang Herr
Jana Albrecht, Eva Distler, Michaela Frey, Shamsul A. Khan, Matthias Theobald, Wolfgang
Herr, Udo F. Hartwig
3rd Dept. of Medicine, Haematology, Internal Oncology & Pneumology, University Medical Centre of Johannes Gutenberg University Mainz, Germany
The graft-versus-leukaemia (GvL) effect after allo-
alleles. In summary, we demonstrate herein that
Specific allogeneic T-cell therapy leading to improved
We observed a significant reduction of human
geneic haematopoietic stem cell transplantation is
AML-reactive CD4 T-cells can be reliably isolated
graft-versus-leukemia effects after adoptive transfer
CD45+ CD33+ AML blasts in bone marrow of mice
mainly mediated by T-cells of donor origin that rec-
and expanded from naive precursors of HLA-DR
requires efficient in vitro generation of leukemia-
engrafted with MZ580-AML one week after trans-
ognize HLA-associated antigens on patient-derived
and –DQ matched healthy donors. Most of them
reactive T-cells and humanized mouse models to
fer of CTL clone 5H11 [median percentage of AML
leukaemia cells. Previous research has mainly
appear to recognize HLA-DP mismatch alleles, in-
analyze the anti-leukemic potential of transferred
blasts: AML only 1.4%; 5H11 0.3%; Melan A CTL
dicating a potentially important role of these alleles
cells in vivo. We therefore recently established an
1.2% (n=5). p-values: AML only to 5H11 p=0.016;
+
focused on CD8 cytotoxic T lymphocytes representing a major part of GvL effector cells. However,
+
+
as targets of the GvL response.
immunodeficient NOD/SCID/IL2Rγc
null
(NSG) mouse
5H11 to Melan A CTL p=0.056; AML only to Melan
CD4 T-cells, commonly regarded as helper cells
model that allows reliable engraftment of human
A CTL p=0.690]. Interestingly, this effect was even
of the adaptive immune system, are also capable
primary acute myeloid leukemia (AML) blasts, par-
more increased in bone marrow, spleen and periph-
of executing direct cytolytic activity. This as well
ticularly those with high-risk FLT3-ITD mutations.
eral blood of recipient mice analyzed four weeks
as the virtual absence of HLA class II expression
Here, we used these mice to analyze the anti-leuke-
post transfer [median percentage of AML blasts in
on non-haematopoietic cells under non-inflamma-
mia effect and homeostasis of human AML-reactive
bone marrow: AML only 87.5%; 5H11 6.4%; Melan
tory conditions makes CD4 T-cells also attractive
cytotoxic T lymphocyte (CTL) clones in two differ-
A CTL 72.4% (n=5). p-values: AML only to 5H11
mediators of the GvL response. Here we sorted
ent AML patient/donor models upon adoptive trans-
p=0.008; 5H11 to Melan A CTL p=0.008; AML only
+
+
+
T-cells from healthy
fer. CTLs were generated from naive CD8 T-cells
to Melan A CTL p=0.032]. In the second model,
donor PBMC by immunomagnetic or flow cytom-
of healthy donors by in vitro stimulation with HLA-
CTL 1E3 and 5B2, generated against MZ653-AML
etry means and stimulated them against primary
class I-identical primary AML blasts and a cytokine
in a HLA-identical setting and cross-reacting with
acute myeloid leukaemia (AML) blasts matched
combination including interleukin (IL)-2/7/12/15/21
MZ667-AML, were transferred into mice engrafted
for HLA-DR and –DQ but not –DP alleles, reflect-
in allogeneic mixed lymphocyte/leukemia cultures
with MZ667-AML. Four weeks following T-cell trans-
ing the common clinical situation. AML-reactive
(Albrecht et al., 2011, Cancer Immunol Immunother).
fer, both CTLs significantly reduced the percentage
CD4 T-cell populations were analysed for cytokine
For AML engraftment, NSG mice (6 to 8 weeks old)
of leukemic blasts in bone marrow of these animals
secretion, HLA restriction, and T-cell receptor Vβ
were sublethally irradiated (150 cGy) and injected
when compared to controls. Ex vivo analyses of CTL
phenotypically naive CD4
+
5
chain usage. Their ability to lyse targeT-cells was
with 5x10 primary AML blasts into the tail vein. To
1E3 and 5B2 re-isolated from spleens one week after
measured in 5h chromium-release assays. In 3 ex-
achieve 1-3% human leukemic blasts in the murine
transfer and restimulated twice in vitro showed per-
tensively studied donor/patient models, leukaemia-
bone marrow, resembling minimal residual disease,
sistent reactivity to AML blasts. Human CD8 CTLs
reactive CD4 T-cell clones could be expanded that
mice were engrafted for 17 to 24 days, depend-
could be detected mainly in bone marrow and spleen
were mainly restricted by HLA-DP alleles according
ing on AML samples used. Subsequently, 5-10x10
6
to antibody blocking experiments. T-cells showed
AML-reactive CTLs cultured for 42 to 56 days were
In summary, we show herein that leukemic burden in
functional properties of Th1 type cells, producing
transfused intravenously three days after the last
NSG mice engrafted with primary human AML blasts
IFN-γ, TNF-α, but not IL-4 upon AML stimulation.
antigen-specific restimulation in vitro. As specificity
can be significantly reduced after a single transfu-
They lysed primary AML blasts as well as patient-
control, AML-engrafted mice receiving Melan A-re-
sion of human AML-reactive CTL clones. To further
derived EBV-B cells at moderate to strong levels (i.e.
active CTLs were included. Mice were supplemented
improve this anti-leukemic effect, experiments with
up to 100% at effector/target ratio of 60/1). More-
with human IL-2, IL7-Fc, and IL-15 in parallel to CTL
repeated CTL transfers are currently ongoing. Our
+
+
+
one week after transfer.
T-cell clones cross-reacted with AML
transfer. IL-2 and IL-15 were additionally injected
model represents a valuable tool to evaluate efficacy
blasts derived from other patients expressing the
three days after transfer. AML infiltration in bone
and homeostasis of in vitro generated leukemia-re-
same single HLA-DP mismatch allele as patienT-
marrow, spleen, and peripheral blood was analyzed
active CTLs in vivo and will be further optimized to
cells used for initial stimulation, suggesting that
by flow cytometry one and four weeks after T-cell
serve as a general platform for testing patient-tailored
these T-cells are directed against disparate HLA-DP
transfer.
T-cell grafts prior to adoptive transfer into humans.
over, CD4
64
3rd Department of Medicine, University Medical Center of Johannes Gutenberg University,
Mainz, Germany
65
016 | Cellular Therapy
017 | Cellular Therapy
Adoptive lymph node-derived HPV-specific T-cell therapy for
cervical cancer
Depletion of naive T-cells using GMP grade CD45RA
­MicroBeads
1
1
1
1
1*
Marij Welters , Zohara Aghai , Vanessa van Ham , Sjoerd van der Burg ,
2
Mariette van Poelgeest 1
1*
1
2
3
1
D. Teschner , E. Distler , D. Wehler , D. Marandiuc , K. Langeveld , M. Theobald ,
1
W. Herr 2
Departments of Clinical Oncology and Gynecology, Leiden University Medical Center, Leiden,
Netherlands.
1
Third Department of Medicine, University Medical Center of Johannes Gutenberg University,
Mainz, Germany
2
Blood Transfusion Center, University Medical Center of Johannes Gutenberg University,
Mainz, Germany
3
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
*
Adoptive transfer of tumor-specific T-cells – which
the LNMC bulk culture procedure for the TDLN of
Alloreactive T-cells are major inducers of graft-
subset showed persistent reactivity to cytomegalo-
have been isolated from blood or tumor and ex vivo
3 patients resulted in similar results as observed
versus-host disease (GvHD) and originate mainly
virus, Epstein-Barr virus, candida, and aspergillus
expanded before infusion into a patient – is a prom-
during the first run. Analyses of the single peptides
from naive precursors. The aim of this study is to
antigens, which could be blocked by HLA antibod-
ising immunotherapeutic approach for the treat-
recognized revealed that the expanded population
establish a good manufacturing practice (GMP)
ies thereby confirming T-cell mediated recognition.
ment of cancer. Activation of tumor-specific T-cells
comprised a polyclonal HPV16-specific T-cell pop-
occurs within the highly specialized microenvi-
ulation. There was no overt expansion of CD25 -
hi
+
ronment of tumor draining lymph nodes (TDLN).
Foxp3 T-cells in the LNMC bulk cultures. Overall,
Previously, we have found that TDLN harbor an-
HPV-specific CD4 T-cells were present in all, but
+
+
+
-
T-cells
Alloreactivity of the CD45RA fraction and the un-
from donor lymphocyte infusion (DLI) products
separated leukapheresis were analyzed in alloge-
as a new approach of primary GvHD prophylax-
neic mixed lymphocyte reaction (MLR) assays.
is. Leukapheresis products from healthy donors
Even in clinically inapplicable HLA mismatch set-
procedure for depleting naive CD45RA
+
tigen-specific T-cells. Therefore, TDLN may be an
one LNMC bulk cultures; CD8 T-cell responses in
(n=6) were separated under clean room conditions
tings (10 out of 10), the number of alloreactive CD8
attractive source for usage in adoptive cell therapy.
2 cultures.
using novel CliniMACS® CD45RA MicroBeads into
T-cells was strongly (around 1-log) reduced upon
+
-
The objectives of the study were to isolate, char-
In conclusion, TDLN represent a rich source of pol-
CD45RA and CD45RA cells during a 4-hour pro-
acterize, and optimally expand human papilloma-
yclonal HPV16 E6- and E7-specific T-cells, which
cedure. Two fractions (i.e. unseparated, CD45RA )
work using single HLA-mismatch antigens had
virus (HPV)-specific T-cells derived from tumor-
can be expanded under GMP conditions for use in
were frozen for subsequent analyses on phenotype
shown significant reduction of alloreactivity in the
draining lymph node mononuclear cells (LNMC)
future trials of adoptive immunotherapy in patients
and function. Flow cytometric assays showed that
CD45RA subset also for CD4 T-cells, alloreactive
for the treatment of patients with HPV16+ end-
with cervical cancer.
depletion by CD45RA beads eliminated >99.9% of
CD4 precursors appeared at similar numbers in
-
+
cells (median 4.4 log depletion, range
stage cervical cancer by the adoptive transfer of
CD45RA
these autologous tumor-specific T-cells.
3.4 - 4.7). The CD45RA subset contained a higher
-
+
CD45RA depletion. While our previous in vitro
-
+
+
both fractions tested in this study when applying
10 out of 10 mismatch MLR. We concluded that in
+
For this purpose LNMC were isolated from TDLN
proportion of CD4
T-cells than the untouched
vitro depletion of CD45RA cells from entire leu-
during radical surgery in patients with cervical
leukapheresis (median 17.5 vs. 11.6 % in separat-
kapheresis products is feasible and highly efficient
cancer. The LNMC were stimulated with HPV16
+
ed viable cells after thawing). In contrast, CD8
using CliniMACS® CD45RA MicroBeads under
E6 and E7 clinical-grade long overlapping peptides
T-cells and CD4 CD25 Foxp3
regulatory T-cells
clean room conditions. The CD45RA target frac-
and clinical-grade available recombinant human
were decreased in the CD45RA fraction (6.8 vs.
-
tion is enriched for memory T-cells and contains
IL-2 for three weeks. Specificity was studied by
+
+
+
+
+
13.9% and 1.3 vs. 1.8%). CD16 CD56 NK cells and
+
-
+
higher proportions of CD4
T-cells, monocytes
proliferation assay, multiparameter flow cytometry,
CD19 B cells were almost completely depleted by
and granulocytes compared to original leukapher-
FoxP3 staining, and cytokine analysis in superna-
CD45RA beads (0.1 vs. 7.7% and 0 vs. 13.9%, re-
esis. In contrast, CD45RA depletion decreases the
+
numbers of CD8 T-cells as well as NK cells, B cells
So far LNMC from 11 patients were studied. The
-
granulocytes were enriched in the CD45RA frac-
and regulatory T-cells to various degrees. T-cell re-
optimal expansion of LNMC cultured with IL-2 re-
tion (40.9 vs. 23.1% and 7.9 vs. 1.6%). CD4 and
+
activity to pathogens appears to be preserved in
tant obtained at day 2 of the proliferation assay.
66
both authors contributed equally
spectively), whereas CD14+ monocytes and CD15
+
+
-
sulted in 30-50 fold increase in cell numbers in 3
CD8 T-cells in the CD45RA- subset were entirely
the CD45RA fraction. Notably, CD45RA depletion
weeks. Almost all cultures showed expansion of
CD45RO-positive and showed very low expression
results in the reduction of T-cell mediated alloreac-
HPV16-specific T-cells as measured by IFN-gamma
of CD62L and CCR7 as well as intermediate ex-
tivity, with apparently stronger efficacy in the CD8
production in the proliferation assay and multipa-
pression of CD27 and CD28, indicating a memory
compared to the CD4 subset.
rameter flow cytometry assay. A complete repeat of
-
phenotype. In IFN-γ ELISpot assays, the CD45RA
67
018 | Cellular Therapy
019 | Cellular Therapy
NK cell subpopulations differ in their tissue specific homing
capacity and impact in GVHD prevention
rhIL-15 and rhIL-2 Enhance the Growth and Function of TCR
Transduced T-cells for Adoptive Immunotherapy
1
1
1
1
1
1
2
1
3
3
3
Kathrin Meinhardt , Ruth Bauer , Irena Kroeger , Julia Schneider , Franziska Ganss ,
2
3
1
1
­Diana Dudziak , Michael Rehli , Andreas Mackensen , Evelyn Ullrich Gina Scurti , Mingli Li , Kelly Moxley , Andre Roy , Heather Embree , Boro Dropulic ,
4
Michael Nishimura 1
Department of Internal Medicine 5, Hematology and Oncology, University of Erlangen-Nürnberg, Erlangen, Germany
1
Loyola University Chicago, Oncology Institute, Cardinal Bernardin Cancer Center, Maywood,
IL, USA
2
Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany
2
3
Department of Hematology & Oncology, University Hospital of Regensburg, Regensburg, Germany
Medical University of South Carolina, Department of Surgery, Center for Cellular Therapy,
Charleston, SC, USA
3
Lentigen Corporation, Gaithersburg, MD, USA
4
Loyola University Medical Center, Department of Surgery, Oncology Institute, Cardinal
­Bernardin Cancer Center, Maywood, IL, USA
Clinical studies exploiting the impact of Natural
+
Infusion of TCR gene modified T-cells for the treat-
sufficienT-cell numbers required for patient treat-
killer (NK) cells during hematopoietic stem cell
plantation in vivo. Interestingly, CD11b NK cells
ment of melanoma has shown promise in recent
ment. Additional culture conditions are being in-
transplantation (HSCT) have provided promis-
migrate to the GVHD target organs, whereas CD27
+
years. However, clinical responses are not com-
vestigated to further enhance the culture process of
ing results. NK cells are a promising tool for cell
NK cells preferentially home to the bone marrow.
parable to responses obtained when using tumor
transduced T-cells for the purpose of gene modified
therapy because of their capacity to lyse tumor cells
Finally, we investigated the role of distinct NK cell
infiltrating lymphocytes (TIL). We hypothesize
T-cell based immunotherapies.
without prior activation and their influence on the
subpopulations in the development of GVHD in
the transduction, culturing and expansion of TCR
innate as well as the adaptive immunity. It is known
a fully MHC mismatched HSCT mouse model. In
transduced T-cells in vitro lead to different charac-
that NK cells are a heterogeneous population and
summary, our comparative study outlines that only
teristics than those found in TIL. Therefore there is
can be divided into functionally distinct NK cell
the CD11b NK cells that migrate to the peripheral
a need to improve upon current culture methods of
subpopulations. Murine NK cells can be separat-
GVHD target organs and provide the most efficient
transduced T-cells which would allow for optimal
ed along their expression of CD27 and CD11b and
cytolytic capacity provide GVHD protection. These
function and growth required for large scale im-
CD117 (c-kit). However, the functional relevance of
new insights are highly relevant for the selection
munotherapy. In an effort to improve upon these
the distinct NK subsets in graft-versus-host-disease
of the optimal NK cell preparation in the field of
culture methods, we determined if adding rhIL-15
(GVHD) has not been investigated in detail so far.
cellular therapy.
to the culture media enhanced growth and/or func-
+
We have established different protocols for isola-
tion of the TCR transduced T-cells.
tion and expansion of murine NK cell subpopula-
TCR transduced T-cells from multiple donors were
tions. These NK subsets were further analyzed in
cultured under several different cytokine condi-
vitro and in vivo in an allogeneic murine GVHD
tions including rhIL-2 at 300IU/mL, rhIL-15 at
model. The four different NK cell subsets provide
100ng/mL or a combination of both at previously
differences in their genomic, phenotypic and func-
stated concentrations. The cells were transduced,
tional properties. Data clearly demonstrate that
cultured and rapidly expanded for a total of approx-
+
CD11b NK cells express multiple genes of cyto-
imately 21 days. Cells were analyzed throughout
toxic pathways and develop the highest tumorlytic
the process for growth, function, phenotype and
capacity. We observed up to 60% tumor lysis by
TCR expression for each of the culture conditions.
-
+
CD27 CD11b
+
NK cells compared to 40-45% by
+
+
-
CD27 CD11b , about 25% by CD27 CD11b and
+
10% by c-kit CD11b- NK cells at an effector-target
+
68
ent sorted NK cell subsets in bone marrow trans-
The results found the cultures which contained
rhIL-15 had a higher production of IFN-γ and greater
TCR expression than the culture conditions which
ratio of 5:1. Interestingly, CD27 NK cells provided
contained rhIL-2 alone. The use of both rhIL-2 and
the highest IFN-γ production upon incubation with
rhIL-15 together in the culture not only enhanced
tumor cells and/or IL-2. We further analyzed the
the function and TCR expression but optimized the
migratory capacity and tissue homing of the differ-
growth of the transduced T-cells which provided
69
020 | Cellular Therapy
021 | Cellular Therapy
Reduced alloreactivity of human memory versus naive
CD8 T-cells in vitro as well as in vivo: Defining optimal
­target-populations for TCR-transfer
Development of clinically implementable imaging strategies for
T-cell receptor-transgenic T-cells
Sebastian Klobuch, Maria Sommer, Matthias Theobald, Ralf Georg Meyer, Wolfgang Herr,
Simone Thomas
Sabine Mall , Calogero D’Alessandria , Sybille Reeder , Annette Frank , Iina Laitinen 3
3
2
1
1
­Michaela Aichler , Axel Walch , Markus Schwaiger , Christian Peschel , Angela Krackhardt Department of Medicine, Hematology, Oncology, and Pneumology, University Medical Center of
the Johannes Gutenberg University, Mainz, Germany
1
Medizinische Klinik III, Klinikum rechts der Isar, Technical University Munich, Germany
2
Nuklearmedizinische Klinik und Poliklinik, Technical Universtiy Munich, Germany
3
Resarch unit Analytical pathology, Helmholtz Zentrum München, Germany
70
1
2
2
2
2
Adoptive transfer of human T lymphocytes is a
CD8 T-cells of naive phenotype. To investigate the
Transfer of T lymphocytes genetically modified
using flow cytometry and immunohistochemistry
promising strategy for treating viral diseases and
relevance of these findings for a clinical application,
with tumor-antigen specific T-cell receptors (TCR)
we could detect a stable engraftment of activated
cancers. However, antigen-specific T-cells cannot
we used a mouse model that allows the analysis of
is an upcoming therapy for cancer treatment.
human peripheral blood mononuclear cells (PBMC)
be generated in every case due to the absence of
T-cell alloreactivity directed against human hemat-
However, clinical trials showed that anti-cancer
in sublethal irradiated Nod/SCID mice after intra-
specific memory T-cells in individual patients or
opoiesis. For this, we reconstituted NOD/SCID/IL-
efficacy of engineered T-lymphocytes needs to be
peritoneal injection. Efficacy of engraftment could
null
+
donors. Grafting T lymphocytes with antigen-spe-
2Rgc
(NSG) mice with human CD34 stem cells
further improved. Better surveillance of adoptively
be enhanced by injection of Interleukin-15.
cific T-cell receptors (TCR) allows the redirection
and adoptively transferred them with naïve and
transferred T-cells in patients requires non-invasive
To monitor TCR-transduced PBMCs in vivo, we
of non-reactive T-cells into tumor- or virus-specific
memory CD8 T-cell populations previously stimu-
and sensitive cell-tracking technologies. High reso-
took advantage of the murinzation of respective
effector T-cells for a broad range of antigens. One
lated againsT-cells of the stem cell donor. In line
lution small animal imaging systems are now in
TCR and used
approach for transfer of TCR is the electroporation
with the in vitro data, TN cells mediated stronger
widespread use, but not all are suitable for clinical
anti-murine TCR antibody for PET imaging. After
of human T lymphocytes with in vitro transcribed
alloreactivity toward donor hematopoiesis than TM
translation. Thus, we aimed to develop a pre-clini-
radioiodination, we could detect specific binding
mRNA, which results in potent effector functions to
cells did. This was shown by a significant decrease
cal in vivo imaging model to monitor and evaluate
to TCR-transduced PBMCs in vitro while viability
+
125
Iodine for radioiodination of an
specific targets for up to one week. Due to the tran-
of human CD45 hematopoietic cells and B-cells in
the efficacy and safety of selected TCR candidates.
and functionality of T-cells remained unaffected.
sient expression of the introduced RNA, a potential
spleen and bone marrow of the mice. Moreover, in
We have previously isolated several allo- and au-
In vivo PET imaging of human TCR-transduced
study protocol would have to include the weekly
vivo proliferation of adoptively transferred T-cells
torestricted TCR specifically recognizing endog-
PBMC engrafted into tumor bearing Nod/SCID mice
administration of TCR redirected T-cells. However,
was mainly detected in mice treated with TN cells
enously presented peptides derived from diverse
using
in an allogeneic setting this approach might be
as measured by BrdU incorporation.
tumor associated antigens presented by MHC class
enhanced signal at the tumor site corresponding to
hampered by the induction of serious graft-versus-
Our data show that human memory CD8 T-cell pop-
I or class II molecules. Transduction of effector
detection of human T-cells within the tumor.
host reactivity through the repeated transfer of
ulations mediate decreased alloreactivity in vitro as
T-cells with these TCR resulted in recognition of
Taken together, we developed a non- invasive
polyclonal donor T-cells. Considering these limita-
well as in vivo. This observation along with strong
hematopoietic and solid tumor cells in vitro. To
imaging model by tracking specifically human
tions, we generated TCR transfected naïve (TN) and
effector function upon TCR transfer makes memory
establish a tumor model suitable to test adoptive
TCR-engineered lymphocytes for further analysis
memory (TM) CD8 T-cell populations, using a CM-
CD8 T-cells promising tools for adoptive immuno-
immunotherapy using these TCR, non-obese dia-
of the efficacy of tumor-reactive TCRs in vivo.
Vpp65-specific TCR as a model antigen receptor.
therapy.
betic/ severe combined immunodeficiency (Nod/
This model will be useful to monitor adoptive
Although both naive and memory T-cell subsets
SCID) mice were inoculated intraperitoneally with
transfer of transgenic T-cells in mice and potential-
showed comparable level of TCR expression upon
a Burkitt´s lymphoma cell line (BJAB). To use one
ly humans and therefore giving important informa-
RNA transfection, CD8 TM cells mediated superior
model for different TCR BJAB cells were eventu-
tion for further optimization of immunotherapeutic
cytotoxicity against CMV-infected targets. In addi-
ally transduced with the respective tumor associ-
approaches.
tion, we analyzed alloreactivity of CD8 TN and TM
ated antigen or MHC restriction elements. Stable
subsets against HLA-mismatched EBV-transformed
tumor development was monitored by positron
B-cells in IFNγ ELIspot and cytotoxicity assays. As
emission tomography/computer tomography (PET/
expected, alloreactivity was mainly mediated by
CT) using 2-[ F]-fluorodeoxyglucose. Furthermore,
124
I-labeled monoclonal antibody showed an
18
71
022 | Cellular Therapy
023 | Cellular Therapy
Adoptive transfer of redirected T-cells targeting a TGFβRII
frameshift mutation frequently occuring in microsatellite
­instable colon cancer
Donor lymphocyte infusion induces polyspecific CD8+ T-cell
responses with concurrent molecular remission in AML with
NPM1 mutation
1
1
1
1
1
1
1
1
1
Else M Inderberg Suso , Sébastien Wälchli , Marit R. Myhre , Weiwen Yang ,
1
1
2
2
1
Sissel Trachsel , Johanna Olweus , Meng Yu , Gunnar Kvalheim , Gustav Gaudernack Susanne Hofmann , Marlies Götz , Cornelia Herbst , Vanessa Schneider , Lu Zhang , Don1
1
2
1
ald Bunjes , Hartmut Döhner , Markus Wiesneth , Jochen Greiner 1
Section for Immunology
1
Department of Internal Medicine III, University of Ulm, Germany
Section for Cell Therapy, Oslo University Hospital-Norwegian Radium Hospital, Oslo, Norway
2
Institute of Clinical Transfusion Medicine, University of Ulm, 89081 Ulm, Germany
2
Adoptive transfer of genetically engineered T-cells
lines harbouring the mutation. Transient TCR ex-
Delayed donor lymphocyte infusion (DLI) already
Survivin, RHAMM, Proteinase 3 and WT-1.
offers great opportunities in cancer immunothera-
pression may also be a safer alternative compared
plays a key role in supporting the graft-versus-
CD8
py. However, recent findings, both in the clinic and
with stable gene transfer for the first evaluation
tumor effect after allogeneic hematopoietic stem
P300, Proteinase 3, Survivin and WT-1 were detect-
in pre-clinical mouse models, indicate that careful
of a novel TCR in the clinic, but requires multiple
cell transplantation (HSCT) although the potency
ed in blood samples after preemptive DLI but not in
consideration of the target antigen should be made
T-cell infusions to compensate for the short-lasting
of DLI differs for each disease entitiy. The graft-
samples before DLI. In parallel, former detectable
to avoid on-target toxicity.
transgene expression.
versus-leukemia (GvL)-effect observed after DLI
NPM1
Safer targets for such T-cell therapy may therefore
A pilot study in a murine model of MSI+ colorec-
is based on the CTL-mediated immunity which is
patient achieved molecular complete remission –
be mutated proteins such as frequently occurring
tal cancer indicated that the colon cancer cell line
reactive against minor histocompatibility antigens
and former mixed chimerism became complete
frameshift mutations resulting in polypeptides
HCT-116 engrafted well and adoptively transferred
(mHAg). To date, the role of leukemia-associated
after DLI.
which are not otherwise available for antigen pro-
redirected T-cells homed to the tumour. Further
antigens (LAAs) in GvL is not clear.
Here, we could demonstrate for the first time poly-
cessing and thus truly tumour specific. The exqui-
studies are ongoing to investigate the in vivo anti-
We therefore analysed peripheral blood of a
specific cytotoxic CD8
site tumour specificity of such mutation also avoids
tumour efficacy and will be reported.
57-year old woman with AML with NPM1 muta-
several known LAAs in a patient with AML
T-cell responses against NPM1-P#1, -P#3,
mut
transcripts were no longer traceable – the
+
T-cell responses against
the problem of low affinity TcRs due to central
tion (NPM1
) who developed molecular relapse
with NPM1mut after preemptive DLI in molecu-
tolerance. One such example is the transforming
without detection of myeloid blasts in the peripher-
lar relapse. Interestingly, the immune responses
growth factor β Receptor II (TGFβRII) frameshift
al blood and bone marrow at d+171 after allo-HSCT
against LAAs were associated with MRD negativ-
mutation found in hereditary non-polyposis colo-
and received preemptive DLI at d+ 260. In parallel
ity. Whether specific cytotoxic T-cell responses
rectal cancers (HNPCC) and around 15% of spo-
to the molecular relapse, chimerism continuously
against epitopes derived from the NPM1
radic colorectal and gastric cancers displaying mi-
decreased to 60 % in the polymorphonuclear and 75
or the polyspecificity of the cytotoxic T-cells against
crosatellite instability (MSI). The -1A mutation in
% in the mononuclear cell fraction. Blood samples
several LAAs are decisive in the elimination of the
an adenine stretch of the TGFβRII gene gives rise to
were taken before and after DLI and investigated
myeloid blasts with NPM1
immunogenic peptides which have previously been
for specific T-cell responses against several LAAs
mined in a larger cohort of patients. All in all it is
used for vaccination of MSI+ colorectal cancer pa-
for which cytotoxic T-cell responses were already
of interest to perform preemptive DLI in patients
tients in a Phase I clinical trial. In one of these
described. In addition, the immune status of the
with molecular relapse systematically. In addition,
patients, we identified and cloned a novel HLA-A2-
patient was determined by measuring the counts
boostering of T-cells against specific LAAs could
restricted TGFβRII frameshift mutation-specific
of CD8 and CD4 T-cells as well as B-cells in the
possibly be an approach to enhance GvL-effect
T-cell receptor (TCR) from a CD8-/CD4- CTL clone.
peripheral blood at several time points. To test spe-
with reducing GvHD-effect at the same time, but
Electroporation of mRNA encoding the TCR into
cific CD8 T-cell responses, ELISpot analysis for in-
the most suitable LAA or combination of LAAs for
polyclonal, in vitro expanded human T-cells
terferon gamma and granzyme B were perfomed.
each disease entitiy still has to be investigated.
+
72
mut
+
+
+
+
+
mut
showed that both CD8 and CD4 T-cells express-
Two epitopes derived from the NPM1
ing the TCR were functional following recognition
(NPM1-P#1 and –P#3) and PRAME (P300 and P435)
of peptide-loaded targets and colon carcinoma cell
were tested as well as one epitope obtained from
mut
mut
peptide
remains to be deter-
peptide
73
024 | Cellular Therapy
025 | Cellular Therapy
In vitro activation of NK cells overcomes resistance of multi­
cellular Ewing sarcoma sphere architecture to NK cell lysis
Animal-component free GMP-grade medium for the activation
and expansion of T-cells
1
1
1
1
Sareetha Kailayangiri , Katharina Leuchte , Bianca Altvater , Simeon Hoffschlag , Jutta
1
1
1
1
1
Meltzer , Sibylle Pscherer , Andrea Luecke , Jenny Potratz , Martina Ahlmann , Heribert
1
1
Juergens , Claudia Rossig Ulrike Kolrep, Nadine Mockel-Tenbrinck, Anne Richter, Hermann Bohnenkamp, Mario Assenmacher, Uwe Odenthal, Veit Bergendahl, and Melanie Fahrendorff
Miltenyi Biotec GmbH, Research & Development, Bergisch-Gladbach, Germany
1
Department of Pediatric Hematology and Oncology, University Children´s Hospital Muenster,
Muenster, Germany
74
Disseminated Ewing sarcoma has remained fatal in
spheres remained more resistant. However, among
Therapeutic applications of T-cells in immuno-
recovery and background stimulation compared to
most patients despite advanced combinatorial treat-
primary tumor cell cultures, consistent differences
therapy have recently gained momentum with the
the use of a standard basal-medium supplemented
ment strategies. Relapses occurring after good initial
in chemosensitivity were not observed between the
promising results in adoptive transfer of antigen-
with 10% human AB-serum. For the automation
response to chemotherapy are caused by residual
two culture systems. Side populations (SP) deter-
specific T-cells for infectious complications after
of such complex procedures, a new cell processing
cells capable to reinitiate and sustain tumor growth.
mined by Hoechst dye exclusion and postulated to
allogeneic stem cell or solid organ transplantation
device was developed. All steps for the CCS pro-
Targeting of these residual cells by cellular immu-
be enriched for tumor initiating cells, were uni-
or for immunotherapy of malignant diseases. Ac-
cessing performed in this fully automated device,
notherapy may sustain remission and improve the
formly absent in monolayers but could be identified
tivation and expansion of these cells for clinical
in a closed system under sterile conditions.
outcome. Specifically, activated NK cells have shown
in three of four VH-64 sphere cultures. However,
application under controlled conditions requires
In conclusion, the newly developed GMP-grade,
promising anti-cancer activity in Ewing sarcoma. To
tumor cells resuspended from spheres did not form
GMP-grade reagents including appropriate antibod-
serum and animal-component-free T-cell medium
explore the value of immunological treatment ap-
subcutaneous tumors in immunodeficient (NOD/
ies, cytokines and media. For these standardized,
demonstrated high lot-to-lot consistency and was
proaches preclinical models are needed that mimic
scid) mice at higher efficiencies than monolayer
reproducible cell cultivation procedures and ex
superior in its performance to other commercially
the anchorage-independent, multicellular growth of
cultures, arguing against higher tumorigenicity of
vivo differentiation, a new serum-free, GMP-grade
available serum-free media. The new medium can
Ewing sarcoma micrometastases.
sphere-cultured cells.
medium (TexMACS GMP Medium) for clinical use
be used to replace human AB-serum supplementa-
Here, we generated Ewing sarcoma spheres from
Long-term coincubation of in vitro activated al-
has been developed. High lot-to-lot consistency
tion for the clinical manufacturing of T-cells result-
four established cell lines (VH-64, TC-32, TC-71,
logeneic NK cells with VH-64 spheres resulted in
has been achieved by eliminating protein com-
ing in easier handling and higher consistency.
A4573,) as well as from three tumor cell cultures
efficient disintegration of spheres and substantial
ponents not relevant for T-cell expansion leaving
In conclusion, the developed GMP-grade and
(MSPES-1, MSPES-4, DC-ES-6) established from
depeletion of viable cells as determined by flow
human serum albumin as the only protein compo-
animal-component free TexMACS GMP Medium
Ewing sarcoma biopsies under serum-free condi-
cytometry. Activated allogeneic NK cells were also
nent. The expansion of T-cells in TexMACS GMP
showed comparable or superior performance to
tions. Standard monolayer cultures were used for
cytolytic against resuspended spheres as well as
Medium upon polyclonal activation using bioti-
other commercially available serum-free media in
comparisons. Phenotypic analysis by flow cytom-
monolayer cultures. This effect was further con-
nylated antibodies against CD2/ CD3/ CD28 loaded
all tested T-cell activation and expansion systems.
etry revealed heterogeneous expression of several
firmed in an autologous system. In short-term co-
on anti-Biotin-MACSiBeads, resulted in expansion
The medium and other relevanT-cell culture com-
surface markers among individual Ewing sarco-
incubation experiments of Ewing sarcoma spheres
rates comparable to what was observed with other
ponents (e.g. antigens, antibodies, cytokines etc.)
mas and between spheres and monolayers. While
(MSPES-4) with autologous NK cells, both intact
commercially available serum-free media. Using
are harmonized with all components of the Clini-
CD133 and the Ewing sarcoma marker CD99 were
spheres, resuspended spheres as well as monolay-
soluble antibodies against CD3 and CD28, more
MACSâ system enabling clinicians to pursue con-
expressed at comparable densities, expression
ers were efficiently lysed.
than 30%-higher expansion rates of viable and
temporary clinical bench to bedside research.
levels of the neural crest marker CD57 (HNK-1) and
Thus, Ewing sarcoma cells cultured under variable
functional T-cells after 6 days of expansion have
of MHC class I proteins were significantly higher in
in vitro conditions differ with regard to phenotype,
been achieved with the new animal-component-
sphere cultures, whereas CD117 (c-kit) expression
function and susceptibility to both chemo- and im-
free medium compared with other serum-free
was lower. Ligands for NK cell activating receptors,
munotherapy. Heterogeneity was also found among
media. Transferring the same protocol to a high
including NKG2D (MICA/B, ULBP1/2/3) as well as
individual cell lines and primary cultures. Activat-
density cell culture system such as a gas permeable
DNAM-1 (CD112, CD155) were expressed at com-
ed allogeneic as well as autologous NK cells effi-
rapid expansion device, high densities of T-cells
parable densities under both culture conditions.
ciently target Ewing sarcoma cells including mul-
with more than 1.5x10 cells/ mL were reached.
Quantitative analysis of the viability of VH-64
ticellular spheres. The sphere model may provide
The generation of antigen-specific T-cells using
cells in the presence of the cytostatic drug doxo-
a useful tool to analyze the contribution of micro-
the Cytokine Capture System IFN-gamma (CCS)
rubicine revealed an increased chemoresistance of
metastatic architecture and serum-free niches to
and the serum and animal-component-free T-cell
spheres compared to monolayers, and resuspended
immune evasion.
medium showed similar results regarding purity,
7
75
026 | Cellular Therapy
027 | Cellular Therapy
Immune-dominant Wilms’ Tumor 1 (WT1) peptide as ­target
structure for cellular immune therapy in acute myeloid
­leukemia (AML)
Establishment of an efficient transient transfection method for
preparation of therapeutics expressing T lymphocytes
Ulrike Buttkereit, Eugenie Hermann, Hellmut Ottinger, Dietrich. W. Beelen
Ruth Eggers, Anja Philippi
Clinic for Bone Marrow Transplantation, University Hospital Essen, Essen, Germany
Cellular Immunotherapy, KIST Europe Forschungsgesellschaft mbH, Saarbrücken, Germany
The “Wilms Tumor Protein 1” (WT1) is a tumor
beadtechnology® was used for enrichment of acti-
Electroporation
antigen expressed in many solid tumors as well
vated T-cells. Patient samples were more sensitive
m(essenger-)RNA provides a powerful tool to in-
IL-2 ER signal sequence.
as in hematologic malignancies such as acute
to stimulation than donor samples.
troduce genetic information non-permanently in
Thus, by varying the moment of transfection, the
myeloid leukemia (AML) and myelodysplastic syn-
Enriched T-cells were expanded in co-culture with
primary T cells.
electroporation device, the instrument settings and
dromes (MDS). WT1 was shown to induce immune
irradiated feeder cells by means of a cytokine cock-
The aim of this study was to establish an efficient
the ER signal sequence we were able to develop
responses in AML patients, e.g. after peptide vac-
tail. After 10 days cultures were restimulated with
mRNA electroporation protocol which enables
an effective mRNA electroporation method which
cination. Furthermore, WT1 expression correlated
autologous PBMC pulsed with WT1- and CMV- Pep-
preparation of therapeutics producing and secret-
allows preparation of pharmaceutical expressing
with the blast percentages in the bone marrow and
Tivator®. Expansion of WT1-specific T-cells was
ing T lymphocytes for cancer therapy. For method
T cells for cancer therapy.
peripheral blood of AML patients. For this reason
possible, however, not as effective as compared
development ex vivo activated donor T cells were
the WT1 protein is a very attractive target for im-
to CMV-specific T-cells. Phenotype of expanded
transfected with in vitro synthesized mRNA coding
munotherapy of AML.
T-cells was of effectormemory type (CD45RA-posi-
for endoplasmic reticulum (ER)-directed eGFP. By
After allogeneic stem cell transplantation the graft
tive, CCR7-negative).
using a BTX-ECM 830 electroporation device set to
versus leukemia effect (GVL) is mediated by donor-
Further characterization and functional analysis of
deliver a single 500 V/800 µs pulse, mRNA transfer
derived T-cells recognizing leukemia-associated
expanded cells are under way.
efficiencies of 80% could be obtained, while neither
antigens such as WT1 in the context of HLA. Adop-
Based on these preliminary data we conclude that
T cell viability nor the cytolytic activity of CD8
tive transfer of antigen-specific T-cells is a novel
the development of a more effective protocol for
T lymphocytes were impaired. We found that the
approach to target residual leukemic cells e.g. after
expansion of WT1-specific T-cells is mandatory.
moment of transfection has a considerable influence
in
vitro
transcribed
on the transfection efficiency, whereas variation of
GMP-conform protocol for generation and ex vivo
the instrument settings (voltage, pulse length) did
expansion of WT1-specific cytotoxic T-cells (CTL).
not improve the results. By pulsing in vitro stimu-
The Streptamer® and Tetramer® technologies were
lated T lymphocytes in an Amaxa-Nucleofector II
cells in the
device, both transfection efficiency and signal in-
peripheral blood of HLA-A2-positive AML patients
tensity could be enhanced. However, with respect
and healthy donors. Very low frequencies of these
to T cell viability and the absolute number of sur-
cells were detected in healthy donors and slightly
viving T cells, the BTX instrument was clearly su-
higher values in AML and in MDS patients.
perior to the Amaxa system. Furthermore we found
To increase WT1-specific T-cells in number, periph-
that the transfection result depends on the choice
eral blood mononuclear cells (PBMC) were pulsed
of the ER signal sequence. Electroporation of acti-
with WT1- PepTivator® for 18h, using CMV-. PepTi-
vated T lymphocytes with mRNAs containing the
used to quantify WT1-specific CD8
eGFP signals than transfer of mRNA containing the
+
relapse. The aim of our project is to establish a
+
76
with
vator® as control. Activation of T-cells was detected
leader sequences of IFN-γ and TGF-β2 resulted in
by expression of CD137 or secretion of IFN-γ. MACS
significantly higher transfection rates and stronger
77
028 | Cellular Therapy
029 | Cellular Therapy
In vivo sunitinib pretreatment improves expansion of tumor
infiltrating lymphocyte from renal cell carcinoma patients
Immunosuppreassive effects of antifungal agents on murine
CD8+ T-cells in vitro and in vivo
1
2
3
1, 3
Aurelie L. Guislain , Hester van Boven , Axel Bex , and Christian U. Blank 1
Division of Immunology
2
Division of Medical Oncology
3
Division of Pathology , Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, A
­ msterdam,
Netherlands
1
2
1
1
Third Department of Medicine, University Medical Center of the Johannes Gutenberg University,
Mainz, Germany
2
Institute of Immunology, University Medical Center of the Johannes Gutenberg University, Mainz,
Germany
3
Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center of the
­Johannes Gutenberg University, Mainz, Germany
Novel targeted therapies achieve objective response
towards NK T-cells, while IFN-production was not
Prophylaxis and therapy of opportunistic invasive
with posaconazole, cell counting showed significant
rates up to 50% in metastatic renal cell carcinoma
different on a per cell level.
fungal infections in immunodeficient patients include
suppression of T-cell proliferation starting at a con-
(RCC), but durable complete remissions are very
Consecutive rapid expansion after culture, using
potent antifungal agents such as posaconazole and
centration of 0.3 µg/ml (mean 6.4 ± SD 1.3x10 cells)
rare. Therefore additional therapeutic options are
anti-CD3/IL-2, induced 100-200 fold expansion
voriconazole. In previous work we could demonstrate
compared to the control group (14.2 ± 0.8x10 cells;
warranted. Adoptive transfer (ACT) of tumor in-
in both groups, expanding preferentially T-cells,
severe inhibitory effects of posaconazole (but not vori+
6
6
3
p=0.002). This effect was confirmed by H-thymidine
filtrating lymphocytes (TIL) has revealed response
allowing a threefold higher final yield when pre-
conazole) on human antigen-specific CD8 T cells in
proliferation assay. Addition of voriconazole at same
rates of up to 70% in melanoma patients and may
treating the tumors with sunitinb.
vitro. With regard to potential effects in patients after
concentrations did not result in any significant in-
lead to consolidation of responses upon sunitinib
Sunitinib in vivo pretreatment improves TIL growth
allogeneic hematopoietic stem cell transplantation, the
hibitory effect on CD8 T-cell proliferation. In pres-
treatment in RCC. Recent data suggest that sunitin-
during the initial culture and does not impair their
aim of the following study is to investigate the immu-
ence of posaconazole, IFN-γ release of alloreactive
ib influences lymphocyte infiltration into the tumor
function or rapid expansion, allowing a higher
nosuppressive effect of posaconazole in vitro as well
CD8 T cells in vitro was significantly lower, compared
+
+
+
as in vivo using a murine model. CD8 T cells were
to T-cells treated with voriconazole or controls (for
cells (DC), and myeloid derived suppressor cells
extracted from spleens of C57BL/6 mice by magnetic
0.3 µg/ml: 64.8 ± 45.8 vs. 209.5 ± 168.4 (voricona-
(MDSC). Furthermore, in vitro assays revealed in-
activated cell sorting. These T-cells were cultured with
zole) vs. 218.0 ± 122.2 (control) spots/2x10 T-cells;
conclusive results on the toxicity of sunitinib on
spleen cells of the filial (F1) generation of C57BL/6 and
p=0.03). In murine in vivo experiments, oral appli-
T-cell function.
BALB/c mice, thus creating haplo-reactive stimula-
cation of 60 mg/kg posaconazole resulted in highest
To address the effect of sunitinib on TILs, we com-
tion in vitro. The antifungal agents posaconazole and
and most stable serum levels (7.1 ± 5.5 µg/ml). After
pared TIL grown from primary tumors pretreated
voriconazole were added in escalating concentrations
transcutaneous OVA-immunization, SIINFEKL-H2-K
with 2 courses sunitinib prior to cytoreductive ne-
(0.3; 1; 3; 10; 30 and 100 µg/ml) to cell culture medium
tetramer staining showed a reduced frequency of OVA-
phrectomy to TIL generated from treatment-naïve
containing 100 U/ml of mIL-2. T-cell proliferation was
specific CD8 T cells in vivo during treatment with
and can impact regulatory T-cells (Treg), dendritic
78
1
Jana Kober , Daniel Teschner , Pamela Stein , René Mönnikes³, Matthias Theobald ,
1, 2
1
1
Markus Radsak , Udo Hartwig , Wolfgang Herr final yield of TIL for ACT studies in RCC.
3
4
b
+
RCC patients. From 6 pretreated primary tumors
measured by cell counting and H-thymidine prolifera-
posaconazole compared to controls (1.1 ± 0.3% vs.
and 6 untreated controls, sterile tumor material
tion assay at days 4, 7, 12, and 14. To analyze allore-
2.6 ± 1.6%; p=0.24). Further flow cytometry analy-
was obtained and digested with collagenase, hyalu-
activity of T-cells in vitro IFN-γ ELISpot assays were
sis upon CSFE labeling revealed an inhibitory effect
ronidase and DNase to obtain tumor single cell sus-
performed on day 12. To establish an in vivo model
of posaconazole on specific lysis of CD8 T cells in
pension. This TIL/tumor suspension was cultured
posaconazole serum levels of mice were measured
vivo (9.8 ± 8.8 vs. 26.6 ± 17.8%; p=0.24). In conclu-
for up to 25 days in 6000IU/ml IL-2 and rapidly
by high-performance liquid chromatography (HPLC)
sion, application of posaconazole shows even stronger
expanded by aCD3/IL2 stimulation afterwards.
after oral administration of 40 mg/kg to 100 mg/kg
inhibitory effects on murine CD8 T cell proliferation
TIL harvested after co-culture with tumor cells
posaconazole once daily for at least 12 days. Subse-
and function than on human T-cells in vitro. Oral ad-
yielded 43 (+/-48) fold expansion from non-pre-
quently, mice were transcutaneously immunized with
ministration of 60mg/kg posaconazole leads to stable
treated tumors versus a 128 (+/-55) fold expansion
the ovalbumin peptide (OVA) to evoke expansion of
serum levels in mice comparable to those reported for
+
+
+
from tumors from patients that had been pretreat-
peptide-specific CD8 T cells in presence of or without
humans, and results in measurable inhibitory effects
ed with sunitinib (p= 0,03). Phenotypic analysis
orally administered posaconazole (60 mg/kg). For de-
on T-cell priming and function in vivo. Further experi-
of the culture product indicated a higher content
+
tecting OVA-specific CD8 T cells and for functional
ments including a murine tumor model are planned to
of NK cells from treatment naïve TIL cultures,
testing, tetramer staining and in vivo analysis of their
investigate the clinical impact of posaconazole-associ-
while sunitinib pretreatment TIL cultures skewed
specific lysis were performed. After in vitro treatment
ated T-cell suppression.
79
030 | Cellular Therapy
031 | Cellular Therapy
A novel process technology for automated NK cell culture and
enrichment
A group of T-cell receptors with strict requirements in the
­hypervariable region, but plasticity in the germline encoded
domains, for recognition of HLA-A2/CD20
Markus Granzin, Mareke Brüning, Iris Spiegel, Sabine Müller, Jessica Blaschke,
Volker ­Huppert, Jürgen Schmitz
Sébastien Wälchli , Lars-Egil Fallang , Weiwen Yang , Even Walseng , Shraddha Kumari ,
2
2
3
4
Harlan Robins , Nishanth Marthandan , Wolfgang Uckert , Mirjam Heemskerk ,
1
Johanna Olweus 1
1
1
1
1
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Section for Immunology, Institute for Cancer Research, Oslo University Hospital-Radiumhospitalet, N-0310 Oslo, Norway
2
Adaptive Biotechnologies, 1551 Eastlake Avenue East, Suite 200, Seattle, WA 98102, USA
3
Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13092 Berlin, Germany
4
Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, Netherlands
Natural killer (NK) cells are a promising tool for
Flow cytometry analysis revealed comparable
We recently published that T-cells specific for allo-
TcR to another resulted in a specificity signature
cancer therapy due to their ability to detect and
expression of CD16, NKp30, NKp46 and NKG2D
geneic HLA-A*02:01 (A2) in complex with a peptide
that was highly similar to the original signatures.
eliminate cancer cells. Using NK cells for therapeu-
for manually or automatically expanded NK cells
from the B cell-restricted protein CD20 (CD20p) can
Taken together, the results demonstrate that the
tic cell transfer in the clinic requires high numbers
either from CD3-depleted or undepleted cultures.
be isolated from HLA-A2 negative donors, with po-
conserved parts (CDR3β and J2-7) of this group of
of these cells. Therefore a method for automated
Results show that large scale automation of NK cell
tential application in immunotherapy of leukemia
TcRs are necessary but not sufficient for recogni-
expansion of human NK cells is developed.
expansion is possible by use of the CliniMACS®
and lymphoma. Interestingly, their T-cell receptor
tion of HLA-A2/CD20p, and furthermore that there
Automation of NK cell expansion was done by use
Prodigy. CD3 depletion of unwanted T and NK like
(TcR) beta chains contained highly biased CDR3
is a high degree of plasticity in the requirements for
of the CliniMACS® Prodigy instrument, a novel tech-
T-cells during culture is feasible.
regions and a conserved J-region, whereas no bias
the surrounding germ line encoded parts.
nology for cell processing in a clinical environment.
could be observed in the use of Vβ domains or
Automated Ficoll density gradient centrifugation was
α-chains. This group of related TcRs could there-
performed to remove erythrocytes and granulocytes
fore be used as a model to test the requirements
from buffy coat samples of healthy donors and to
for the germline versus the hypervariable regions
obtain peripheral blood mononuclear cells (PBMC).
for target recognition. We first showed that the
This automated method yielded a suitable number
previously reported sequence bias could be con-
of PBMC and a better depletion of granulocytes
firmed by deep sequencing. Next, a group of TcRs
compared to the manual PBMC preparation. PBMC
that contained the conserved CDR3 motif and J2-7
were cultivated with IL-2 and OKT-3 leading to an
in their β-chains, but highly variable sequences in
expansion of NK cells, but also of NK like T-cells and
the remaining parts, were expressed and charac-
8
80
1
T-cells. NK cell numbers up to 5,1x10 were reached
terized according to fine specificity. We found that
with expansion factors of 54-208-fold after three
any manipulation of the CDR3β or J2-7 sequences
weeks. NK cell purities of 5-52% were obtained.
negatively affected the TcR binding. Together, these
Pure NK preparations are preferred with regard to
results demonstrated dependency on the conserved
analysis of clinical outcome and to reduce the risk
parts for HLA-A2/CD20p recognition. When scru-
of graft versus host disease possibly caused by con-
tinizing the TCRs further we found that five of the
taminating T-cells.
TCRβ-chains retained the ability to bind tetramers
Removing co-cultured T and NK like T-cells by
and produce IL-2 when combined with an α-chain
CD3-depletion during the expansion phase was
from another CD20 specific TcR. Interestingly, for
possible and resulted in a higher purity of NK cells
some of the swapped TCRs the fine specificity was
(56 - 96%). The cells retained their proliferative
different compared to the original TcRs, suggest-
capacity after depletion and expansion factors of
ing a possible alteration in the antigen-binding
30-156-fold were obtained.
region. In contrast, swapping the CDR3β from one
81
032 | Cellular Therapy
033 | Cellular Therapy
Analysis and optimization of a p53264-272-tumor antigen-­specific
­single chain TCR in terms of TCR mispairing in human T-cells
Effect of cord blood regulatory T-cells on natural killer cell
­differentiation and function
1
1
1
1
Diana Knies , Beate Hauptrock , Hakim Echchannaoui , Edite Antunes , Simone Thom1
1
2
1
3
as ­­, Sebastian Klobuch , Philippe Guillaume , Wolfgang Herr , Pedro Romero , Matthias
1
1
­Theobald , Ralf-Holger Voss 1
Department of Hematology & Oncology, University Medical Center, Mainz, Germany
2
Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland
3
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne
branch, Hôpital Orthopédique, Lausanne, Switzerland
82
1, 2
1, 2
1, 2
1, 2
Isabela Pedroza-Pacheco , Martha Luevano , Richard Duggleby , Alejandro Madrigal 1, 2
and Aurore Saudemont
1
Anthony Nolan Research Institute, Royal Free Hospital, London NW3 2QG, UK
2
UCL Cancer Institute, University College London, Royal Free Campus, London NW3 2QG, UK The adoptive transfer of tumor reactive T-cell recep-
facilitating the association to any TCRα chain. We
Regulatory T-cells (Tregs) may offer a promising
cell-mediated GvL is adversely impacted by CB Treg
tor (TCR) reprogrammed human T-cells represents an
observed a higher incidence of mispairing in Jurkat-
treatment for graft versus host disease, the main
therapy.
efficient strategy to increase the GVL (graft-versus-
76 T-cells compared with human T-cells due to a less
complication of haematopoietic stem cell trans-
leukemia) effect after allogeneic stem cell transplan-
competitive situation between exogenous and human
plantation (HSCT). To assess the feasibility of this
tation. A safeguard in terms of mispairing between
TCRα/β chains. IFNγ secretion-assays after stimula-
therapy, it is important to consider their interaction
the endogenous and exogenous TCR chains that likely
tion with p53264-272-peptide loaded K562-A2 targeT-cells
with natural killer (NK) cells, which are described
results in neoreactivities against self antigens and thus,
elicited decreased IFN-γ production reflecting higher
as key effector cells of graft versus leukaemia (GvL).
autoimmune pathologies, would be the use of a single
mispairing and less expression of the p53264-272-specific
Several groups have studied NK cell suppression by
chain (sc) TCR construct. We asked ourselves whether
scTCR. We speculated that the linkage of both mole-
Tregs. However, a better understanding of this in-
a single chain TCR scaffold of the domain order Vα-
cules on a single plasmid via a 2A-element would kinet-
teraction using cord blood (CB) as a cell source is
Linker-Vβ-Cβ entirely prevents pairing with arbitrary
ically favor the association of the nascently produced
necessary, since CB is being increasingly used as a
TCRα-chains due to sterical hindrance. For this, we
chains in the endoplasmic reticulum. Here we observed
source of stem cells for HSCT.
are using 2 different retroviral murine p53264-272-tumor
less but still residual mispairing with various human
This study examined CB cells as a model of trans-
antigen- specific scTCR constructs. The first one is re-
or murine TCRα chains. The scTCR p53 revealed more
plantation and focused on the effect of Tregs on (i)
ferred to as a 2-retroviral vector system because of the
potency to interact with murine than human TCRα
NK cell phenotype and cytotoxicity and (ii) NK cell
cloning of the p53264-272-tumor antigen-specific scTCR
chains, thus the amount of interaction correlated with
differentiation from CD34 stem cells to mature NK
and the accessory murine (Mu) Ca-domain on sepa-
the competitive strength of the respective TCRα chain.
cells during a 5-week culture. Phenotype and via-
rate retroviral vector backbones. Mu Ca is essential for
In line with that, IFNγ secretion-assays after stimula-
bility (7AAD-Annexin V) were assessed by flow cy-
the stable expression of the p53264-272- tumor antigen-
tion with p53264-272 peptide loaded K562-A2 targeT-cells
tometry and NK cell killing capacity by Cr-release
specific scTCR. The second one links both molecules
proved less IFNγ-secretion for a mispaired scTCR p53.
assay. To assess Treg effects on different stages of
on a single retroviral vector by a self-splicing 2A-
It is important to note that even tiny amounts of mis-
differentiation, Tregs were added at 1:4 ratio with
element and accounts for their concomitant expres-
paired scTCRs may end up with clonal expansion after
NK cells at 2, 9, 16, 23 and 30 days of culture. When
sion. We performed pairing analysis of the murine
neoreactive antigen encounter and potential autoreac-
Tregs were added at day 16, there was a significant
p53264-272-tumor antigen-specific scTCR with various
tive T-cells due to the large proliferative capacity of
decrease in the percentage of committed iNK cells
human (­­­gp100280-288­, ­AML14-22) as well as murine
T-cells in general. Therefore we aimed at improving
(median, 27.16% NK cells v. 6.24% NK cells with
(p53264-272, MDM281-88) TCR alpha chains (TCRα) of dif-
the stability of the Vα/Vβ-domains by protein design.
Tregs, p <0.01) and CD56
ferent antigen-specificities and variable Va-subfamilies
The optimized p53264-272-tumor antigen- specific scTCR
29.05% NK cells v. 4.78% NK cells with Tregs, p
mimicking any endogenous TCRα chain. The analysis
demonstrated an equal efficacy and specificity towards
<0.05); yet NK cell killing capacity was not af-
was done in human T-cells, which equals a competi-
the cognate antigen when compared to the unmodified
fected (p>0.05). Similarly, whilst isolated NK cells
tive situation in the presence of endogenous TCRs as
scTCR p53 while entirely preventing residual mispair-
co-cultured with Tregs decreased the expression
well as in Jurkat-76 lacking endogenous TCRs, which
ing in vitro. Notably, the modification led to a slightly
of activating receptors (CD16, NKG2D, NKp46 and
equals a non-competitive situation. In a 2-retroviral
increased TCR affinity as shown by tetramer satura-
DNAM-1), this effect was not maintained.
vector system the analysis of TCR surface expression
tion binding and Scatchard analysis. In conclusion, mi-
So far, no persistent effect of Tregs on NK cell dif-
indicated some mispairing of the scTCR p53 with any
spairing analyses emphasizes the need for both retrovi-
ferentiation and function has been detected; there-
TCRα chain. We hypothesize that the Vα/β-domains
ral vector as well as TCR protein design to safely apply
fore, a complete study with activated Tregs is re-
of the scTCR p53 may have a propensity to dissociate
them in adoptive immunotherapy of cancer.
quired. This study may help to determine if NK
+
51
bright
NK cells (median,
83
034 | Cellular Therapy
035 | Cellular Therapy
An optimized single chain p53(264-272)-specific T-cell receptor
devoid of ON/OFF target autoimmunity in a humanized mouse
model of adoptive T-cell transfer
Identification of donor-derived antigen-specific CTLs for clinical
application using cysteine modified tetramers
Jutta Petschenka, Edite Antunes, Matthias Theobald, Hakim Echchannaoui
Cathrine F Knetter, Nadia Mensali, Weiwen Yang, Glenn Buene, Lars-Egil Fallang,
Even Walseng, Reidulf Stray Pedersen, Johanna Olweus
Department of Internal Medicine III (Hematology, Oncology, Pneumology), University Medical
Center Mainz, Germany
Several studies have demonstrated the clinical effi-
off-target autoimmunity, an optimized single chain
We have recently demonstrated that CTLs recog-
pensive and only available as off-the-shelf reagents
cacy of adoptive T-cell therapy for targeting cancer.
(sc.) p53 TCR was engineered. Mice receiving op-
nizing a peptide from the B cell specific antigen
for known epitopes, preventing testing of large
Using HLA-A2.1 transgenic mice, we have demon-
timized sc. p53 TCR-transduced T-cells under con-
CD20 (CD20p) in context of foreign HLA-A*0201,
numbers of potential new targets. An alternative is
strated the feasibility of T-cell receptor (TCR) gene
ditions that promote expansion of the adoptively
efficiently and specifically kill patient-derived
to generate pHLA multimers from an in-house pre-
transfer into T-cells to circumvent self-tolerance to
transferred T-cells did not show any sign of GvHD.
malignant B cells. Such CTLs could potentially
cursor of the HLA-allele and a UV-sensitive peptide
the widely expressed human p53 (264-272) tumor-
Because the expression of p53 antigen on normal
be isolated from healthy donors and used to treat
by the method developed by Toebes, Shumacher
associated antigen and developed approaches to
tissues raises the concern of potential on-target tox-
patients with malignant lymphoma and leukemia.
and colleagues (Nat.Med. 2006). Briefly, a stable
generate high-affinity CD8-independent TCR.
icity, we performed adoptive T-cell transfer experi-
In the setting of standard allogeneic hematopoietic
HLA class I complexed with a photolabile epitope is
However, a particular safety concern in TCR gene
ments in humanized mice expressing the human
stem cell transplantation, a mismatch on one HLA
UV irradiated in the presence of a specific peptide,
transfer is the formation of mixed TCR dimers
p53 protein (Hupki mice) and did not observe any
allele is permitted. Here, three separate donor - re-
which will rescue the complex from degradation.
between introduced and endogenous TCR chains,
sign of TCR gene transfer-associated GvHD in this
cipient pairs were simulated by utilizing healthy
Although providing a high degree of flexibility, a
resulting in the potential generation of self-reac-
model. In addition, we could show that optimized
donors mismatched on HLA-A*0201, but otherwise
limitation with this approach might be low stabil-
tive T-cells. Therefore, strategies to favor matched
sc. p53 (264-272)-specific TCR redirected T-cells
allelic identical when typed for the A, B, C, DRB1
ity, and that peptides with low affinity do not ef-
TCR chains pairing and thus enhancing TCR cell
mediate antitumor reactivity in a mouse tumor
and DQB1 HLA loci. The donors were harvested
ficiently replace the UV-sensitive peptide residues.
surface expression, including optimization of TCR
model.
by leukapheresis. Monocytes from the HLA-A*0201
To improve the peptide exchange of such peptides,
encoding nucleotide sequences, introduction of an
In conclusion, these mouse studies show that adop-
positive donor were used to generate dendritic cells
an alternative is to utilize a system where modi-
additional inter-chain disulfide bond between the
tive TCR gene transfer-associated GvHD does not
(DCs). By day 8, the DCs were pulsed with CD20p
fied HLA multimers are generated by introducing
TCR α and β chain constant domains, coexpression
occur with the optimized sc. p53 (264-272)-specific
and used to stimulate T-cells from the HLA-A*0201
a cysteine residue in the heavy chain. In addition,
of both TCR α and β encoding-genes using self-
TCR therefore suggesting that sc. p53 TCR may rep-
negative donor. The T-cells were re-stimulated by
a GC sequence is added to the N-terminal part of
cleaving 2A virus peptide-based retroviral vectors
resent a new and safe approach for TCR-based gene
day 19 with HLA-A*0201 positive CD20p-pulsed
the peptide of interest, hence allowing a cysteine
and murinization of human TCR constructs have
therapy of p53-associated malignancies.
EBV-LCL from a third party donor. Add posi
bridge to be formed between the modified heavy
been applied.
HLA-A*0201/CD20p –specific T-cells can be iden-
chain and peptide.
Nevertheless, adoptive transfer of mouse T-cells
tified and isolated using staining with soluble
We tested multimers presenting the CD20p gener-
transduced with optimized p53 specific TCRs into
HLA-peptide (pHLA) multimers, and have the po-
ated by different approaches for binding to a cell
p53-deficient humanized (A2Kb) mice was associ-
tential to selectively cause graft-versus-leukemia.
line transduced with a CD20 specific TCR. The
ated with the development of lethal autoimmunity
On day 19, CD8 positive multimer reactive T-cells
cysteine modified multimers had been stored at
due to the formation of self-reactive TCRs infiltrating
were identified from all three HLA-A*0201 nega-
4°C for 20 days, and showed an increased stability
vital organs, such as spleen, liver and bone marrow.
tive donors. The same principle could be used to
as compared to 1) an in-house multimer generated
+
In this mouse model, we have evidence that CD4
generate T-cells specific for other cell-type specific
by refolding HLA-A*0201 with the wt CD20 peptide
T-cells play a key role in controlling the develop-
peptides presented on foreign HLA. However, when
or 2) a commercial multimer, both stored at equal
ment of graft-versus-host disease (GvHD) as we
selecting the specific T-cells it is critical to avoid
conditions, or 3) a multimer generated by the UV
could show that the onset of the pathology is ac-
the contamination of allo-reactive T-cells with the
exchange procedure, as measured by a higher frac-
celerated in mice receiving only genetically modi-
potential to cause graft-versus-host disease. There-
tion of multimer positive cells and a higher mean
+
84
Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radium­
hospitalet, Oslo, Norway.
fied CD8 T-cells, compared to mice receiving both
fore, pHLA multimers that are highly stable and
fluorescenc intensity. This method could potentially
T-cell subsets.
give a bright staining of the specific T-cells is es-
improve the isolation of highly specific T-cells able
To further prevent TCR gene transfer-associated
sential. Commercially available tetramers are ex-
to induce graft-versus-leukemia for clinical use.
85
036 | Cellular Therapy
037 | Cellular Therapy
Reprogramming T-cells with an optimized Melanoma-specific human single chain T-cell receptor results in substantial tumor cell
recognition but also in mispairing with endogenous TCR chains
Targeting dendritic cells with functionalized nanoparticles
1
1
1
1
2
1
1
1
2
3
1
1
Beate Hauptrock , Martina Teresa Glomski , Diana Knies , Edite Antunes , Pedro Romero ,
1
1
Matthias Theobald , Ralf-Holger Voss M. Sommer , P. Okwieka , O. Zupke , M. Diken , G. Baier , D. Wolff , E. Distler ,
1
3
1
1, 3
1
M. Theobald , K. Landfester , W. Herr , V. Mailänder , R. G. Meyer 1
Department of Hematology & Oncology, Johannes Gutenberg University of Mainz, III. Medical
Clinic & Policlinic, Building for R&D, Obere Zahlbacherstrasse 63, Mainz, Germany
1
Department of Hematology, Oncology and Pneumology and 2Institute for Translational
­Oncology and Immunology, University Medical Center Mainz, Germany
2
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne
branch, Hôpital Orthopédique, Lausanne, Switzerland
3
Max Planck Institute for Polymer Research, Mainz, Germany
86
Adoptive transfer of gene-modified T-cells with
morantigen p53 cannot exclude mispairing of the
Acute GvHD is a major cause of morbidity after al-
tive studies revealed that the relative uptake of NP
tumorantigen-specific TCRs is a strategy to induce
scTCR with a panel of irrelevant TCRα chains. To
logeneic hematopoietic stem cell transplantation
in the tissues was higher than in PB.
tumor regression in cancer patients. However, trans-
study if the optimized human scTCR/Cα-construct
(HSCT). It is mediated by donor T-cells activated
To investigate the ability of targeting DC in vivo, we
fer of double chain (dc)TCRs into human T-cells
also mispairs with TCRα chains of other specifici-
by host antigen-presenting cells (APC). Even T-cell
performed first experiments in a humanized NSG
contains the risk of forming hybrid dimers with the
ties, TCR-deficient Jurkat-76 cells were transduced
depletion does not completely prevent acute GvHD.
mouse model. Mice that had been humanized with
endogenous TCR chains. Here, we used a human
with different combinations of the scTCR and full
Thus, manipulation/depletion of APC may be an
1x10 human peripheral blood stem cells (PBSC)
single chain (sc)TCR specific for the Melanoma-
length TCRα chains. Expression of the gp100-
alternative for preventing allo-reactivity. We iden-
successfully engrafted human CD45-positive cells
antigen gp100 by linking the variable Vα-domain
specific scTCR was determined by flow cytometry
tified polystyrene-based polymeric nanoparticles
as well as DC subsets in bone marrow (BM), spleen
to the TCR β-chain as a potential tool to prevent
using a vβ14-specific antibody. The minimally
(NP) that labeled dendritic cells (DC) in vitro. The
and PB. We injected the NP to these mice intrave-
TCR mispairing. After retroviral co-transduction of
murinized scTCR was expressed in JurkaT-cells in
used NP (diameter 80 - 160nm) were functionalized
nously and analyzed them as outlined. The fluo-
this human scTCR and the constant domain of the
combination with Cα or with different human and
with either NH3- or COOH-groups to target DC.
rescence pattern of humanized mice did not differ
TCR α-chain (Cα) into human T-cells no expression
murine TCRα chains, indicating that mispairing
NP were tested by FACS analysis in regard to
from that of the non-humanized controls. After 4
was detectable at the cell surface. Expression was
takes place. However, low MFI of vβ14-staining in
their internalization by and potential toxicity on
days, 14.4% of spleen cells were positive for human
restored when the human C-domains were replaced
6
JurkaT-cells transduced with the scTCR/TCRα in
monocyte-derived DC. Additionally, intracellu-
CD45 with CD14-positive monocytes being almost
by murine C-domains. Since murine elements
comparison to scTCR/Cα led to the conclusion that
lar localization of NP was confirmed by confocal
completely NP-positive (92.2%). When analyzed
might be immunogenic in patients, we aimed at
only few mispaired scTCR/TCRα-dimers reached
Laser Scanning Microscopy (cLSM). Incubation of
by laser scanning microscope (LSM), the particles
altering only single amino acids. We and others de-
the cell surface. After co-incubation with gp100
monocyte-derived DC with these NP had no impact
were attached to the outer cell membrane.
termined a lysine in murine Cβ as a pivotal residue
peptide-loaded T2-cells, JurkaT-cells harbouring
on viability on DC. Moreover, unlabeled as well as
In summary, we identified polymeric NP for la-
for expression of human TCRs. Recently, 9 murine
mispaired scTCR/TCRα produced no IFNγ, dem-
NP-labeled DC exhibited same surface marker ex-
beling DC in vitro without a distinct influence on
amino acids being responsible for expression and
onstrating that the specificity of the scTCR was
pression profiles (e.g. CD86 and HLA-DR) and thus
phenotype or function of the cells. Different NP
functionality when introduced into human dcTCRs
lost by mispairing with an irrelevant TCRα chain.
showed no differences in their function as antigen
showed distinct bio-distribution patterns in vivo
were identified by Sommermeyer et al. (JI 2010).
Similar experiments performed in human T-cells
presenting cells.
when applied systemically to NSG-mice. The bio-
These amino acids were introduced into the gp100-
showed only a moderate expression of the scTCR
For in vivo analyses, the NP have been triple-loaded
distribution was not altered when mice were hu-
specific scTCR/Cα. Contrary to the published data,
when combined with an irrelevant TCR α chain,
with BODIPY (FACS), IRDye-780 (in vivo biofluo-
manized. Still, NP were not taken up by DC in vivo.
we only detected a moderate enhancement in scT-
probably due to favourable pairing of the full length
rescence imaging, BFI), as well as platinum (tissue
We currently investigate the influence of serum
CR-expression of T-cells transduced with the mini-
TCRα chain with endogenous TCR.
distribution). NP were injected to 5 to 12 weeks
protein on NP uptake. In addition, more specific
null
(NSG) mice intravenously. BFI
mally murinized scTCR/Cα. To improve stability,
In summary, our new constructs represent promis-
old NOD/SCID/γc
additional disulfide bonds were introduced. This
ing candidates for adoptive T-cell transfer because a
directly after i.v. injection showed a fluorescence
led to a substantial increase of expression, cytokine
substantial antitumor effect is provided. Mispairing
peak in the liver. At later time points, fluorescence
production and lysis of peptide loaded targeT-cells
with endogenous TCR chains cannot be excluded,
redistributed also to regions of lung, salivary
and melanomas by T-cells bearing the minimally
but expression levels of hybrid TCRs are low so that
glands, and spleen. After 4 days, mice were sac-
murinized scTCR/Cα.
substantial so-called “off-target”-effects are unlikely
rificed and the fluorescence of organs and tissues
Our recent data showed that the use of a murine
to occur. However, it is desirable to develop methods
was measured. Particles were found in peripheral
scTCR/Cα with the specificity for the universal tu-
to prevent residual TCR mispairing
blood (PB), liver, lung, spleen and skin. Compara-
surface-modifications (e.g. Fc-fragments, specific
antibodies) are being evaluated.
87
038 | Cellular Therapy
039 | Cellular Therapy
CD8+ T-cell-specific transfer of TCR genes enhances tumor cell
killing
Next Generation Sequencing of γδ T-cell receptor repertoires
1
1
2
3
1
Qi Zhou , Irene Schneider , Inan Edes , Annemarie Honegger , Patricia Bach ,
4
5
4
1
2
Kurt Schönfeld , Axel Schambach , Winfried Wels , Sabrina Kneissl , Wolfgang Uckert ,
1
and Christian ­Buchholz 1
Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
2
Max-Delbrück-Center for Molecular Medicine and Humboldt-University Berlin, Institute of
­Biology, Berlin, Germany
3
Department of Biochemistry, University of Zürich, Zürich, Switzerland
4
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany
5
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
88
1, 2
1, 2
1
1, 2
1
Tana Omokoko , Lisa Rüssel , Martin Löwer , Petra Simon , Andrea Breitkreuz ,
1
1
1
1, 2
Valesca Boisguerin , Katja Manninen , John Castle and Ugur Sahn 1
TRON gGmbH, Translational Oncology at the University Medical Center, Johannes Gutenberg
University Mainz, Germany
2
UniCell GmbH, Kupferbergterrasse 17-19, Mainz, Germany
Transfer of tumor-specific T-cell receptor (TCR)
molecules may stimulate effector functions of the
γδ T-cells are promising agents for cancer im-
genes into patient T-cells is an attractive approach
T-cells. The data suggest that CD8-LV represents a
munotherapy since they have the capability to
transfer of ex vivo activated Vγ9Vδ2 T-cells.
in cancer immunotherapy. Currently available
powerful novel vector type for TCR gene therapy
release inflammatory cytokines (IFNγ, TNFα) and
Beyond that the isolation of high-affinity Vγ9Vδ2
vectors deliver genes into all types of lymphocytes
and other applications in therapy and basic re-
chemokines and exert direct and indirect cytotoxic
TCRs may allow for effective cancer-specific redi-
not only cytotoxic T-cells. We describe here a novel
search requiring CD8-positive T-cell specific modi-
activity against tumor cells, either alone or in as-
rection of αβ T lymphocytes via TCR gene transfer
CD8 targeted lentiviral vector (CD8-LV), which de-
fication.
several clinical trials: in vivo activation or adoptive
sociation with other innate and adaptive immune
thereby circumventing the problems associated
livers genes exclusively and specifically to CD8-
effector cells such as NK cells and cytotoxic T lym-
with αβ TCRs being MHC restricted and usually of
positive cells. The targeting ligand displayed on the
phocytes. Additionally they are able of eliciting
low affinity due to the nature of peptide self anti-
vector surface is a single-chain variable fragment
antibody-dependenT-cell cytotoxicity (ADCC).
gens as well as mispairing with endogenous α or
(scFv) derived from the CD8-specific monoclonal
About 1 - 5 % of human T lymphocytes express γδ
β TCR chains, which could lead to autoimmunity.
antibody OKT8. CD8-LV mediated stable reporter
T-cell receptors (TCRs). The major γδ T-cell subset
With that in mind we used the Illumina HiSeq 2000
gene transfer, both, in vitro in human peripheral
residing in the peripheral blood of healthy adults
sequencing system for detailed Vγ9Vδ2 T-cell reper-
blood mononuclear cells, and in vivo in human-
expresses Vγ9Vδ2 TCRs, which respond to nonpep-
toire analysis ex vivo and after stimulation with ZA.
ized mice. This vector efficiently transferred genes
tidic prenyl pyrophosphate metabolites, referred to
We also employed a novel technology platform
encoding TCRs recognizing the melanoma-reactive
as phosphoantigens (pAgs) in a major histocompat-
for the isolation and validation of functional TCR
antigen tyrosinase. Strikingly, T-cells genetically
ibility complex (MHC)-independent manner.
genes from single T lymphocytes for the isolation
modified with CD8-LV killed melanoma cells re-
pAgs are produced by virtually all living cells. Most
of high-affinity Vγ9Vδ2 TCRs from the same donor
producibly more efficiently than CD8-positive cells
importantly, isopentenyl pyrophosphate (IPP) the
and correlated the data looking for characteristics
transduced with a conventional lentiviral vector
most common pAg, which is produced through the
of specific clonotypes.
(VSVG-LV). TCR surface density, expression levels
mevalonate pathway, can activate Vγ9Vδ2 T-cells at
of cytokines such as IFN-γ and TNF-α, as well as the
concentrations that are found in tumor cells but not
proliferative activity of the cells were excluded as
in healthy tissues. Aminobisphosphonates (NBP)
being causative. However, CD8-LV transduced ef-
can be used to induce controlled IPP accumulation
fector cells contained a small fraction of cells with
in antigen presenting cells and tumor cells to ac-
a relatively increased CD8-expression level on their
tivate Vγ9Vδ2 T-cells. We and others have shown
surface. Since CD8 functions as co-receptor for TCR
that zoledronic acid (ZA) the most potent NBP cur-
recognition of tumor cell, we propose that CD8-LV
rently available for clinical use, can be used for ef-
transduction enhances CD8 expression thus result-
ficient activation of Vγ9Vδ2 T-cells when combined
ing in enhanced sensitivity of TCR recognition and
with low doses of IL-2. This paved the way for the
anti-tumoral activity. Moreover, multiple contacts
development of two strategies for the treatment
between the scFv on the vector surface and CD8
of cancer patients that are currently evaluated in
89
040 | Cellular Therapy
041 | Cellular Therapy
In vitro “on-target”-reactivity of affinity-modified
p53264-272 ­tumor antigen-specific TCRs retrovirally transduced
into ­human ­T-cells
Cellular and molecular events controlling acquisition of
­cytotoxic activity by tumour-reactive CD4+ T-cells during
­melanoma progression and immunotherapy
1
1
1
1
1
Karolina Mroz , Lukas Eckhard , Diana Knies , Simone Thomas , Sebastian Klobuch ,
2
3
1
1
­Philippe Guillaume , Pedro Romero , Matthias Theobald , Ralf-Holger Voss Katharina Bergerhoff, Frederick Arce, Karl Peggs, Sergio Quezada
Cancer immunology unit, University College London, UCL Cancer institute, London, UK
1
Department of Hematology, Oncology, and Pneumology, Universal Medical Center of Johannes Gutenberg University, Mainz, Germany
2
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1005 Lausanne­/
Epalinges, Switzerland
3
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne
Branch, Hôpital Orthopédique, Lausanne, Switzerland
CD4
antigen of the wild type sequence 264-272 was de-
In order to study potential “on-target”-reactivity in vitro
orchestrator compartment of the adaptive immune
veloped in HLA-A2-transgenic mice and proved to
we chose autologous A2 human T-cells as targets for
response while CD8
T-cells are considered the
Furthermore, a microarray was performed in order
recognize its cognate antigen with high affinity in
TCR p53-retrovirally reprogrammed T-cells. The target
effectors that execute direct cytotoxicity against
to determine the differences in gene expression
the subnanomolar range in a MHC-A2-restricted and
T-cells were activated by either CD3/CD28 magnetic
cancer cells. However, recent work illustrated the
between naïve, tolerant, normal helper and the
CD8-independent manner. However, from literature it
beads or OKT3, which would mimick the in vivo situ-
significance of the understudied tumour reactive
tumour reactive CD4 cells in the peripheral lymph
end up with proliferative exhaustion and activation
+
ation after cognate antigen recognition by adoptively
transferred TCR p53-redirected T-cells. We observed very
+
T-cells have mainly been presented as the
but accounts for a higher plasticity of the cytotoxic
dogenously processes and presents the p53264-272 antigen.
is known that high-affinity TCRs might prematurely
+
+
CD4 T-cells during immunotherapy and tumour
+
+
+
CD4 cells including CD8 atypical pathways.
+
nodes and the tumour itself. The analysis was par-
progression. Using the CD4 Trp1 transgenic (Tg)
ticularly focused on the lineage specific differen-
induced cell death (AICD) which likely compromise
low (5-15%) but reproducible amounts of A2 target rec-
melanoma mouse model and multicolour flow cy-
tiation markers and transcription factors but also
an effective immune response in vivo. Additionally,
ognition above background for different PBMC donors in
tometry, the molecular and cellular mechanisms
on the up- or down regulation of genes that might
“cross-reactivity” against irrelevant self-antigens and
12-hours 51-chromium release assays. “On-target”-cyto-
+
that underlie the function of these cytotoxic CD4
play a role in the tumour microenvironment, for
“on-target”-reactivity directed against basal levels of
toxicity correlated to the effector functionality of the wild
T-cells are sought to be elucidated. For this, the
example of transmembrane proteins and receptors.
+
cognate self-antigen presentation on normal cells might
type and TCR p53 mutants as estimated from peptide
tumour reactive CD4 cells are tracked and exam-
Amongst others, the chemokine receptors CCR2
lead to autoimmune pathologies. Hence, we prompted
titration. A mock control for various responders did not
ined in a lymphodepleted, tumour-bearing host
and CCR5 were shown to be significantly upregu-
us whether by moderately decreasing the affinity of the
elicit any comparable A2-restricted antigen recognition in
which undergoes immunotherapy. Adoptive trans-
lated on cytolytic CD4 cells in the tumour, which
TCR p53 within a narrow tenfold range the longevity
all experiments and alloreactivity could not be detected
+
-
of p53-specific T-cells may be improved and potential
in different A2 /A2 -mixed target/responder lymphocyte
“on-target”-toxicity may be reduced while sustaining
cytotoxicity assays. Intriguingly, A2 responders bearing
efficient recognition of tumor cells. We focused on Asp
-
+
any of the TCR p53 variants recognized A2 targets with
+
+
emphasises the important role of the tumour micro-
which lack expression of the melanoma differentia-
enviroment on the differentiation of the cytotoxic
tion antigen (tyrosinase-related protein 1 [TRP1]),
phenotype.
into wild type B6 mice results in the recognition
+
higher efficacy than A2 responders. The sensitivity of
of TRP1 by the CD4 Trp1Tg in the host and their
may be dominantly involved in antigen recognition. A
the A2-restricted and antigen-dependent recognition was
acquisition of cytotoxicity.
vast array of amino acid exchanges were introduced
increased by using the HLA-A2-crosslinking antibody
The analysis of lineage specific differentiation
by site-directed mutagenesis and rapidly screened for
BB7.2 which led to substantial higher amounts of specific
markers and transcription factors on the cytotoxic
+
CD4 cells surprisingly led to the impression of a
-
+
expression and effector function by RNA transfer into
antigen recognition. Specific lysis of A2 targets by TCR
human T-cells. We identified TCRα S116C, S116G, S116T,
p53-reprogrammed autologous and A2 responders was
mixed lineage phenotype: the CD8 specific tran-
and S116A-bearing TCR p53 mutants with gradually
diminished by pulsing them with the competitive A2-
scription factor Runx3 was highly expressed in
lower affinity in tetramer analysis, and effector function
high-affine peptide FluM1. A2 targets were barely lysed
the isolated CD4
(IFNγ-secretion and cytotoxicity). Subcloned retroviral
at all which further supports the specificity of the ob-
(GzmB), the cytolytic enzyme responsible for the
TCR p53 constructs showed similar expression and ef-
served “on-target”-reactivity towards A2 T-cells. Since
cytotoxicity of CD8 cells. However, the CD4 cells
fector functionality in T-cells. However, although in
this mechanism referred to as “fratricide” might attenu-
lacked at the same time the expression of Eomes,
tetramer analysis the S116T and S116A TCR p53-deriva-
ate the effectiveness of adoptive immunotherapy we are
the direct inducer for GzmB expression in CD8
tives proved to be weak, they were almost as efficient as
currently developing molecular strategies to prevent this
cells. This novel expression pattern clarifies that
the wild type in peptide titrated effector function and in
adverse reaction in the human T-cell compartment.
the differentiation of the ‘killer’ phenotypes is not
-
+
+
fer of CD4 cells from naïve Trp1 transgenic mice,
115 and Ser 116 which lie on the tip of CDR3α and thus,
recognition of the osteosarcoma Saos-2/143 which en90
+
The TCR p53 directed against the tumor-associated
+
+
cells, along with Granzyme B
+
+
+
+
a complete reprogramming to the CD8
lineage
91
042 | Cellular Therapy
043 | Cellular Therapy
Generation of MCSP-specific, MHC-independent T-cells by RNA
electroporation at a clinically feasible scale
Reprogramming bulk CD8+ and γ/δ T lymphocytes with a specificity for adenovirus by electroporation of TCR-encoding mRNA
1
2
2
1
Christian Krug , Nicole Hoffmann , Patrick Schmidt , Katrin Birkholz , Beatrice Schuler1
1
2
1 *
1 *
Thurner , Gerold Schuler , Hinrich Abken , Jan Dörrie , , and Niels Schaft ,
1
Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany
2
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
*
Share senior authorship
* 1
* 2
1
1
Jan Dörrie , , René Geyeregger , , Ina Müller , Niels Schaft 1
Department of Dermatology, RNA-group, Universitätsklinikum Erlangen, Erlangen, Germany
2
St. Anna Children’s Cancer Research Institute, Clinical Cell Biology and FACS Core Unit, Vienna,
Austria
*
These authors contributed equally
Adoptive T-cell transfer has proven to be a prom-
anti-CD28 antibody to obtain sufficient numbers
An allogenic haematopoietic stem cell transplant
As reprogrammed T-cells still express their en-
ising approach for the immunotherapy of cancer
of T-cells for clinical application. The transfected
(HSCT) combined with chemo-and radiotherapy
dogenous TCR, they may be alloreactive and
during the last decades. The clinical application
T-cells were checked for receptor mediated T-cell
can be the last resort in treating chemotherapy-
might cause graft versus host disease (GvhD).
of such therapies was held back by several prob-
activation and cryopreserved. After thawing, the
resistant hematopoietic malignancies in children.
γ/δ T-cells, in contrast, are not alloreactive, and
lems so far, as tumor-infiltrating lymphocytes (TIL)
cells were again tested for cytokine secretion and
The time to immunological engraftment of the
these cells would be good tools to prevent allore-
were difficult to obtain. In addition, the transfer
cytolytic capacity upon exposure to tumor cells.
transplant in these patients can take up to several
activity against recipienT-cells if the donor is not
of wild-type T-cell-receptors (TCR) into autologous
Finally we performed in vivo experiments in mice
weeks or even months. During this time there is
completely matched. Therefore, we investigated
T-cells was hampered by the naturally weak an-
to test the functionality of CAR-modified T-cells.
often no protection against adenoviral (ADV) infec-
whether γ/δ T-cells could also be reprogrammed
tigen-affinity of those receptors and the dysfunc-
Healthy donor- and patient-derived T-cells could
tion, which causes substantial transplant-associat-
by α/β TCR-RNA electroporation. TCR-transfected
tional MHC-restricted antigen-presentation by the
be expanded by the optimized protocol in a way
ed death. The use of effective antiviral drugs such
γ/δ T-cells also produced IL-2, TNF, and IFNγ in
tumor. Engineering T-cells with chimeric antigen
that is feasible under GMP-conditions and yields
as Ganciclovir or Cidofovir is limited and associ-
response to peptide-loaded targeT-cells. Since most
receptors (CAR) provides an elegant solution for
enough T-cells for the intended clinical application.
ated with significant side effects, and it results in
γ/δ T-cells are CD8-negative, and the introduced
those problems. CAR feature an antibody-derived
The engineered T-cells produced IL-2, TNF, and
prophylactic overtreatment in a large proportion of
TCR might benefit from CD8 co-binding, we co-
binding domain (scFv) which is fused to signaling
IFNγ after stimulation with MCSP+ tumor cells,
individuals. An alternative effective therapy is the
electroporated mRNA coding for CD8 molecules.
domains of the T-cell-receptor complex. After engi-
and this ability was maintained after freezing and
adoptive transfer of ADV-specific T-cells. However,
These TCR-CD8-co-transfected γ/δ T-cells showed
neering with a CAR, the T-cells are able to recog-
thawing. Furthermore, CAR-modified T-cells lysed
10 to 20 % of all HSCT donors lack ADV-specific
an increased cytokine production. In a direct com-
nize the native, unprocessed antigen on the tumor
tumor cells in an antigen-specific manner after cry-
T-cells. In this case, a solution could be the repro-
parison between TCR-transfected CD8 α/β T-cells
cell surface. As CAR T-cells may mediate autoim-
opreservation. Moreover, first in vivo experiments
gramming of donor T-cells with an ADV specificity
and TCR-CD8-co-transfected γ/δ T-cells we found
mune reactions, we decided to modify T-cells by
showed that the transfected T-cells prolonged the
by transfer of a TCR.
that γ/δ T-cells produced more TNF and IFNγ, but
RNA-electroporation in order to achieve transient
survival of immunodeficient mice after co-injection
In this study, we transfected CD8
T-cells with
less IL-2 in response to antigen. In addition, the
CAR expression and thereby eliminating the risk
with tumor cells.
mRNA encoding an HLA-A1-restricted, ADV-spe-
TCR-transfected γ/δ T-cells also efficiently lysed
of permanently ongoing autoreactivity.
Taken together the study provides a clinically ap-
cific TCR by electroporation. The TCR-transfected
peptide-loaded targeT-cells. Surprisingly, the co-
In this study we electroporated T-cells from healthy
plicable protocol for the generation of antigen-spe-
CD8 T-cells specifically recognized peptide-load-
+
+
+
+
transfection of CD8 molecules did not improve the
donors or late-stage melanoma patients with mRNA
cific T-cells in a transient manner for use in the
ed HLA-A1 dendritic cells (DC) and tumor cells
cytolytic capacity of γ/δ T-cells.
encoding different CAR molecules which recognize
immunotherapy of cancer, therefore avoiding some
and responded with secretion of the pro-inflamma-
Taken together, we show here for the first time that
the melanoma-associated chondroitin sulfate prote-
of the safety concerns that are associated with per-
tory cytokines IL-2, TNF, and IFNγ. Furthermore,
not only α/β T-cells but also γ/δ T-cells can be re-
oglycan (MCSP), which is strongly expressed on the
manent modification of T-cells.
TCR-transfected CD8
+
T-cells were also able to
programmed with an ADV specificity by TCR-RNA
surface of almost all melanomas but shows limited
lyse peptide-loaded targeT-cells. Most importantly,
electroporation. These cells may be good tools in a
expression on healthy tissues. T-cells were ex-
TCR-transfected CD8 T-cells also recognized ADV-
new strategy for the immunotherapy of ADV infec-
panded prior to electroporation by stimulation with
infected targeT-cells in an antigen-specific manner.
tion after HSCT.
+
the agonistic anti-CD3 antibody OKT3 and/or the
92
93
044 | Improving Immunity
Immunotherapeutic potential of fully human anti-Macrophage
Migration Inhibitory Factor antibodies in different mouse
cancer models
Enhancing Immunity
1
2
3
1
Patrice Douillard , Thorsten Hagemann , Michael Freissmuth , Michael Thiele ,
1
1
1
1
Dirk Völkel , Hans Peter Schwarz , Fritz Scheiflinger , Randolf J Kerschbaumer
1
Baxter Innovations GmbH, Vienna, Austria.
2
Barts Cancer Institute, Queen Mary University of London, UK.
3
Institute of Pharmacology, Medical University Vienna, Austria.
Macrophage migration inhibitory factor (MIF) is
pro-inflammatory cytokines within the tumor, (iii)
known as a cytokine exhibiting a broad range of
reduce angiogenesis and (iv) prolong the survival
immune and inflammatory activities. The involve-
of the tumor-bearing mice. Similarly, the antibod-
ment of MIF in inflammation, autoimmune dis-
ies were further shown to significantly suppress
orders and in tumorigenesis makes it a potential
tumor growth in xenograft models of ovarian and
target for therapeutic intervention. MIF is up-reg-
prostate cancer.
ulated in a large variety of human neoplasms (e.g.
In conclusion, our data suggest that our fully
pancreatic, breast, prostate, colon, brain, and lung
human anti-MIF antibodies have a good potential
tumors) and several studies report that MIF expres-
for clinical use in oncology. The data are in line
sion closely correlates with tumor aggressiveness
with the role of MIF as a promoter of tumor growth
and metastatic potential. MIF’s biological activities
and the concept of MIF neutralization as a potential
have been shown to contribute to tumor growth
strategy to inhibit tumor progression.
and metastasis by different mechanisms: (i) MIF
activates cell signaling cascades leading to tumor
suppressor down-regulation and enhanced proliferation; (ii) MIF induces angiogenesis and tumor
invasiveness by controlling hypoxic adaptation and
induction of proangiogenic factors such as VEGF;
(iii) as a pro-inflammatory cytokine MIF is a key
mediator of tumor micro-inflammation.
Baxter has developed fully human antibodies that
efficiently bind and neutralize MIF. According to
their potential to neutralize biological MIF activities in vitro, a subset of antibodies was tested in
nude mouse xenograft models for prostate, ovarian
and pancreatic cancer. Human cells were implanted orthotopically into the pancreatic cancer
model and the anti-MIF antibodies were injected
7 days after tumor establishment. The anti-MIF
94
antibodies were able to (i) suppress tumor growth
and metastasis (ii) reduce the production of host
95
045 | Improving Immunity
046 | Improving Immunity
Antibody fusion proteins of cytokines and costimulatory
­ligands for cancer immunotherapy
Single-chain bispecific antibodies activate human regulatory
T-cells and trigger their suppressive function
Vanessa Kermer, Nora Hornig, Roland Kontermann & Dafne Müller
Stefanie Koristka , Marc Cartellieri , Anke Theil , Claudia Arndt , Anja Feldmann , Irene
1
3
3
4
4
Michalk , Katrin Töpfer , Achim Temme , Martin Bornhäuser , Gerhard Ehninger , Marc
1
1, 2
Schmitz , Michael Bachmann Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany
1
2
1
1
1
Institute of Immunology, Medical Faculty ‘Carl Gustav Carus’, Technical University Dresden,
Fetscherstr. 74, 01307 Dresden, Germany
2
DFG-Center for Regenerative Therapies Dresden, Technical University Dresden, Fetscherstr.
105, 01307 Dresden, Germany
3
Department of Neurosurgery, University Hospital ‘Carl Gustav Carus’ Dresden, Fiedlerstr. 23,
01307 Dresden, Germany
4
Medical Clinic and Policlinic I, University Hospital ‘Carl Gustav Carus’ Dresden, Fetscherstr. 74, 01307 Dresden, Germany
+
Cytokines of the common cytokine receptor γ-chain
observed for the antibody-4-1BBL fusion protein.
Bispecific antibodies (bsAb) were designed for the
tempted to cross-link Tregs to PSCA prostate cancer
family and costimulatory members of the B7- and
Thus, the development of antibody fusion proteins
redirection of cytotoxic immune cells against cancer
cells. This proved to be possible as the incubation of
TNF-family have shown great potential to support
with cytokines and costimulatory ligands provides
cells and hold great potential for the immunotherapy
Tregs together with PC3-PSCA cells in the presence
the generation and development of an antitumor
promising options for cancer immunotherapy.
of malignant disorders. In general, a single-chain
of a bsAb CD3xPSCA leads to the upregulation of
immune response. In order to improve the efficacy
bsAb molecule consists of the variable heavy and
various activation markers as well as to the release
of such molecules at the tumor site we designed
light chains of two monoclonal Abs connected via
of the immunomodulatory cytokine IL-10. Moreover,
novel antibody fusion proteins for therapeutic ap-
flexible linker peptides. This facilitates the simul-
bsAb-redirected Tregs showed a remarkable capac-
proaches, focusing either on optimized presenta-
taneous binding of two different antigens, e.g. the
ity to attenuate proliferation and cytokine secretion
tion or a combined mode of action. Thus, we in-
activating CD3 complex on T-cells and a tumor as-
of cocultured autologous CD4
vestigated the possibility to improve the efficiency
sociated antigen on tumor cells. Thereby, T effector
The suppressive potency of bsAb-activated Tregs
of IL-15 presentation in a targeted approach by
(Teff) cells can be cross-linked and activated against
was further verified in vivo. Administering Tregs
the incorporation of an IL-15Rα-chain fragment,
a targeT-cell subsequently leading to the efficient
together with PC3-PSCA tumor cells and a bsAb
mimicking
+
Teff cells in vitro.
presentation.
killing of the latter. The feasibility and efficacy of
CD3xPSCA abrogated the anti-tumor effect of coin-
Therefore, an antibody-cytokine fusion protein
bsAb for tumor treatment has been demonstrated
jected CD4 Teff cells and promoted tumor growth
was generated composed of an antibody moiety
not only in vitro and in several mice studies but also
in athymic nude mice.
targeting the tumor stromal fibroblast activation
in first clinical trials. However, so far it has not been
In conclusion, our findings provide first evidence
protein (FAP), an extended IL-15Rαsushi domain
investigated whether regulatory T-cells (Tregs),
that bsAb can efficiently activate Tregs against a
and IL-15. Enhanced proliferation and cytotoxicity
which also carry the CD3 antigen on their surface,
surface antigen in a TCR independent manner, and
of T-cells in vitro as well as an improved antitu-
get activated by bsAb in a similar way as their Teff
trigger their suppressor function both in vitro and
mor effect in a tumor mouse model in vivo were
cell counterparts.
in vivo. In view of these results, the potential risk
achieved. For a combinatorial approach, antibody
Among various immune escape mechanisms, the re-
of Treg activation should be taken into considera-
fusion proteins with the costimulatory ligands B7
cruitment of Tregs to the tumor microenvironment
tion when applying bsAb for cancer treatment. On
and 4-1BBL, directed against different antigens
plays an important role. Tregs accumulated in tumor
the other hand, given their indispensible role in es-
(FAP and endoglin) on the same targeT-cell were
tissues limit anti-tumor responses and promote
tablishing and maintaining peripheral tolerance, an
generated, displaying costimulatory activity in a
tumor progression. Consequently, increased Treg
antigen- and/or site-specific recruitment of Tregs
target-dependent manner. In combination with a
numbers correlate with poor survival prognosis of
into inflamed tissues using bsAb may offer novel
bispecific antibody (FAPxCD3) enhanced activa-
the affected patients. In light of this, there is an
therapeutic options for Graft versus Host Disease,
tion and proliferation of PBMC was shown, that
urgent need to evaluate whether Tregs will be acti-
transplant rejection or autoimmune diseases.
could be further increased by the application of
vated by bsAb within the scope of a tumor therapy.
both costimuli. Strongest costimulatory effect on
Using a recently described bsAb directed against
physiological
trans
+
proliferation and cytotoxicity of CD8 T-cells was
96
1
CD3 and the prostate stem cell antigen (PSCA) we at-
+
Koristka, S. et al. (2012) J. Immunol. 188: 1551-1558.
Koristka, S. et al. (2012) Ms in preparation.
97
047 | Improving Immunity
048 | Improving Immunity
Immunomodulation of peripheral blood mononuclear cells by
PHA and irradiated K562
Heterologous adeno-poxvirus combination for immunogenic
cancer targeting
1 2
3
4
5
6
Abdolkarim Sheikhi - , Hedyeh Banaei , Nasrin Yahaghi , Karim Saadati , D Robert Siemens 1
Immunology Dept., Dezful Faculty of Medical Sciences, Dezful, Khuzestan, Iran
2
Immunology Dept., Ahwaz University of Medical Sciences, Ahwaz, Khuzestan, Iran
3
Allameh High School, Shiraz, Fars, Iran
4
Payam-noor University, Zanjan, Iran
5
6
Surgery Dept, Valie Asr Hospital, Zanjan University of Medical Sciences, Zanjan, Iran
Anatomy and Cell Biology Dept., Queens University, Kingston, Ontario, Canada
1
1
1
1, 2
Markus Vähä-Koskela , Marko Ahonen , Noora Rouvinen-Lagerström , Anna Kanerva , Fang
3
1
2
4
5
1
Zhao , Sari Pesonen , Päivi Pakarinen , Jarmo Salo , Vincenzo Cerullo and Akseli Hemminki 1
Cancer Gene Therapy Group, Molecular Cancer Biology Research Program, Transplantation Laboratory, Haartman Institute and Finnish Institute of Molecular Medicine, University of Helsinki, Finland
2
Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland
3
Advanced Microscopy Unit, Haartman Institute, University of Helsinki, Finland
4
Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Finland
5
Immunovirotherapy Group, Molecular Cancer Biology Research Program, University of Helsinki, Finland
Objective: Natural killer (NK) cells are an impor-
Background: Cancer therapy has traditionally been
of intratumoral NK cells than in any other tested
tant subset of cytotoxic lymphocytes and are best
limited by acquired cellular resistance and immu-
regimen. Switching viruses also led to a decrease
characterized by their ability to spontaneously kill
nosuppression. In this regard, oncolytic viruses
of neutralizing antibodies in the serum at study
virally infected and tumor cells but the mechanisms
make promising therapeutics as they interfere
endpoint compared to repeated administration of
contributing to deficient NK activity in patients
with multiple cellular pathways and elicit inflam-
the same virus.
with cancer remains unclear. NKp44 and NKG2D
mation, which also promotes anti-tumor immune
Conclusions: Heterologous combination therapy in-
are of the main NK activating receptors involved in
responses. However, innate and adaptive responses
creased anti-tumor efficacy in an adenovirus resist-
recognition and killing of tumors. Here we studied
are typically biased toward foreign (viral) antigens
ant tumor model, suggesting molecular synergy to
the stimulatory effects of K562 on induction of
rather than self-derived tumor antigens. In order to
overcome innate tumor defenses. In immunocom-
NKp44 and NKG2D expression on human PBMCs.
circumvent this problem, we have tested sequential
petent hosts, combination virotherapy activated
Materials and Methods: The NK activity of PBMCs
combination of two powerful yet mechanistically
innate and adaptive immune responses that corre-
against DU-145 was determined with 51Cr-release
distinct oncolytic viruses: adenovirus and vaccinia
lated with reduced tumor burden without increas-
assay. The PBMCs were stimulated with PHA, on 3
virus. The combination presents several theoreti-
ing anti-virus immunity. These results demonstrate
occasions (3-PHA-PBMC) and were incubated with
cal synergies on the molecular and immunological
several benefits of heterologous combination viro-
irradiated K562 (iK562). The expression of CD56,
levels.
therapy that warrant further studies to support
NKG2D and NKp44 were detected with reverse
Methods: The feasibility of combining oncolytic
clinical translation.
transcription-PCR and flowcytometry. Results: PHA
vaccinia virus with adenovirus to overcome innate
+
stimulation increased the proportion of CD56 cells
tumor defenses was tested in vitro and in a dis-
and up-regulated NKG2D and NKp44 expression.
seminated intraperitoneal human ovarian cancer
Co-incubation with iK562 didn‘t change NKG2D
xenograft model in SCID mice shown previously to
but increased NKp44 expression. NK activity of
acquire resistance to human adenovirus [Liikanen
3-PHA-PBMC after co-incubation with iK562 was
et al., 2011, Mol Ther]. Immunological impact of
significantly increased.
sequential virus therapy was assessed in C57 black
Discussion and Conclusion: Our results demon-
mice carrying subcutaneous B16.OVA tumors.
strated that the mitogen and iK562 exposure to
Results: Combination with vaccinia virus provided
PBMCs can significantly improve NK activity which
significant therapeutic benefit in an adenovirus-
is related to the higher expression of NKp44 and
resistant tumor model. In immunocompetent mice,
NKG2D. These data may help to improve cancer
intratumoral injection of 1e8 PFU of vaccinia virus
immunotherapy protocols.
followed six days later by 2e10 VPs of oncolytic adenovirus seemed to provide the greatest therapeutic effect and was associated with greater induction
98
99
049 | Improving Immunity
050 | Improving Immunity
Immunotherapeutic synergy between anti-CD137 mAb and
­intratumoral admistration of a cytopathic Semliki Forest virus
encoding IL-12
Antitumoral responses against liver implanted tumors induced
by Semliki Forest virus expressing IL-12 can be potentiated by
coadministration of IL-15
1
1
1
2
José I. Quetglas , Juan Dubrot , Jaione Bezunartea , Miguel F. Sanmamed ,
1
1, 2
1
Sandra Hervas-Stubbs , Ignacio Melero , Cristian Smerdou 1
2
Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA),
­University of Navarra, Pamplona, Navarra, Spain
Medical Oncology Department, Clínica Universidad de Navarra, Pamplona, Spain
Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Navarra, Spain.
Semliki Forest virus (SFV) expression vectors
the agonist antibody. An additional effect of the
We have previously shown that recombinant
non-treated animals showing profound changes in
contain a single positive strand RNA genome and
combinatorial treatment was a marked reduction of
Semliki Forest virus (SFV) vector expressing in-
lymphoid and myeloid immune populations at day
are able to express high levels of heterologous pro-
humoral responses against SFV particles, reaching
terleukin-12 (SFV-IL12) had potent antitumoral ef-
4 after treatment. Interestingly, a marked increase
teins in many differenT-cell types, including tumor
levels that would allow efficient readministration
ficacy in a subcutaneous model of murine MC38
of IL-15Rα expression was observed on tumor spe-
cells. It has previously shown by our group that
of the viral vector. This efficacious combinatorial
colon adenocarcinoma, leading to complete tumor
cific CD8(+) T-cells of animals that responded to
intratumoral injection of a SFV vector encoding
immunotherapy strategy offers feasibility for clini-
regressions in >90% of animals when they were
therapy compared to non-responders. Since IL-15
IL-12 (SFV-IL-12) combines high expression of IL-12
cal translation since anti-CD137 mAbs are already
treated intratumorally with 10 viral particles (vp).
has been associated with memory immune re-
with induction of stressfull apoptosis in infected
undergoing clinical trials and development of clin-
However, when MC38 tumors were implanted in
sponses these results support the idea that estab-
malignanT-cells. Agonist antibodies directed to the
ical-grade SFV-IL-12 vectors is in progress.
the liver, an organ where colon cancer usually
lishment of this type of response is important for
costimulatory receptor CD137 (4-1BB) can strongly
metastasize, SFV-IL-12 efficacy was reduced to
tumor elimination. Accordingly the combination
amplify pre-existing cellular immune responses
<50% complete regressions. This indicates that
of SFV-IL-12 with recombinant IL-15 was able to
towards weak tumor antigens. In this study we
antitumoral responses are greatly dependent on the
increase the antitumoral effect of the vector leading
demonstrate that a combined strategy consisting
tissue where the tumor develops. We reasoned that
to a significant increase in survival in comparison
of intratumoral injection of SFV-IL-12 and systemic
characterization of antitumoral immune responses
to mice treated only with SFV-IL12 or IL-15. These
delivery of agonist anti-CD137 mAb provides pow-
against intrahepatic tumors could provide useful
data strongly supports the hypothesis that antitu-
erful synergistic antitumoral effects against poorly
information for the design of more potent antitu-
moral effects of SFV-IL-12 against tumors in the
immunogenic B16 melanomas (B16-OVA and B16.
moral strategies against liver tumors. Treatment of
liver could be potentiated by stimulating memory
F10) and TC-1 lung carcinomas. Effector CD8 β
+
liver MC38 tumors with 10 vp of SFV-IL-12 induced
T-cells were sufficient to mediate complete tumor
a high population of tumor specific CD8(+)CD62L(-)­
eradications. Accordingly, there was an intense
effector T lymphocytes in all animals at 7-10 days
synergistic in vivo enhancement of CTL-mediated
after vector inoculation. However, this effector
immunity against tumor antigens OVA and TRP-2.
phenotype appeared earlier in animals responding
This train of phenomena led to long-lasting tumor-
to therapy. All treated mice showed high levels of
specific immunity against rechallenge, attained
functional specific CD8(+) T-cells at 8 days post-
transient control of the progression of concomi-
treatment by both in vivo killing and IFNγ ELISPOT
tant tumor lesions that were not directly treated
assays, but these levels decreased along time, being
with SFV-IL-12, and caused autoimmune vitiligo.
this reduction more pronounced in animals that
Importantly, we found that SFV-IL-12 intratumoral
had not been able to eliminate tumors. The role of
injection induces high expression of CD137 on most
CD8(+) T-cells in tumor elimination was confirmed
+
8
8
T lymphocytes, thereby
by depletion studies. Infiltration studies were per-
providing more abundant targets for the action of
formed in liver, spleen and tumors of treated and
tumor infiltrating CD8
100
José I. Quetglas, Marta Ruiz-Guillén, Juan R. Rodríguez-Madoz, Jaione Bezunartea,
Erkuden Casales, José Medina-Echeverz, Sandra Hervás-Stubbs, Pedro Berraondo, Jesús
Prieto, and Cristian Smerdou
immune responses.
101
051 | Improving Immunity
052 | Improving Immunity
Tumor-specific CD4+ T-cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory
T-cells
CpG-ODN induced upregulation of BTLA mediating selective
inhibition of human B cells
1
1
1
2
1, 2,3, 4*
5, 6*
1, 2,3, 4
Ilseyar Akhmetzyanova , Gennadiy Zelinskyy , Simone Schimmer , Tim Sparwasser 1
and Ulf Dittmer Marie-Laure Thibult
, Jean-Paul Rivals , Emilie Mamessier , Julie Gertner1, 2,3, 4
1, 2,3, 4
5
1, 2,3, 4*
7*
Dardenne , Sonia Pastor , Daniel E. Speiser , Daniel Olive and Laurent Derré 1
Institute for Virology of the University Clinics in Essen, University of Duisburg-Essen,
45147 Essen, Germany
1
INSERM U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, F-13009, France
2
Aix-Marseille Université, UMR 891, F-13284, Marseille, France
Institute for Infection Immunology, TWINCORE, Feodor-Lynen-Str. 7, 30625 Hannover, Germany
3
Institut Paoli Calmettes, IBiSA Cancer Immunomonitoring Platform, F-13009, Marseille, France
4
CNRS, UMR7258, centre de recherche en Cancérologie de Marseille, Marseille, F-13009, France
5
Clinical Tumor Biology and Immunotherapy Unit, Ludwig Center of the University of L
­ ausanne,
Switzerland
6
University Hospital Center and University of Lausanne (CHUV), Switzerland.
7
Urology Research Unit, Department of Urology, Lausanne University Hospital (CHUV), Switzerland.
*
M.L.T, J.P.R., D.O. and L.D. contributed equally to this work.
2
The important role of tumor-specific cytotoxic
B cells activation requires several signals via the BCR
protozoal DNA, which can be mimicked by synthetic
CD8 T-cells (CTLs) is well-defined in the immune
upon antigen binding, and via various co-activating
oligodeoxynucleotides, such as CpG motifs. Stimula-
+
control of the tumors but the role of effector CD4
and inhibitory receptors, mostly members of the B7/
tion of TLR9 by CpG motifs initiates the intracellular
T-cells is poorly understood. In the current re-
CD28 co-receptors family. These molecules regulate
MyD88-mediated signaling pathway, resulting in the
search we have used a murine retrovirus-induced
numerous checkpoints of immune cells functions,
release of pro-inflammatory cytokines, and plasma-
tumor cell line of C57BL/6 mouse origin, namely
regulating differentiation, maturation, adhesion,
cytoid differentiation, promoting B cell proliferation,
FBL-3 cells, as a model to study basic mechanisms
chemotaxis and the release of soluble factors. Several
class switch recombination and antibody produc-
of immunological control and escape during tumor
co-inhibitory receptors have been identified and the
tion. In this study we investigated the role of BTLA
formation. This study shows that tumor-specific
therapeutic blockade of these molecules is in prom-
in human B cells. We show that BTLA expression is
CD4 T-cells are able to protect against virus-in-
ising clinical development. The recently described
modulated during B lymphocyte differentiation, with
duced tumor cells. We show here that there is an
inhibitory receptor B and T lymphocyte attenuator
an enhanced expression in IgM memory and transi-
+
+
+
expansion of tumor-specific CD4 T-cells produc-
(BTLA, CD272) is structurally and functionally related
tional B cells. Then, we analyzed BTLA expression by
ing cytokines and cytotoxic molecule granzyme B
to CTLA-4 and PD-1, and is expressed by the major-
B cells in vaccinated melanoma patients. When CpG
in the early phase of tumor growth. Importantly,
ity of lymphocytes. Interestingly, most BTLA studies
were used as adjuvant for vaccination, we observed
we demonstrate that in vivo depletion of Tregs and
were realized on T lymphocytes. Interaction of BTLA
a sustained expression of BTLA, whereas in absence
CD8 T-cells in FBL-3-bearing DEREG transgenic
with its ligand Herpes Virus Entry Mediator (HVEM,
of CpG, a progressive downregulation of BTLA was
mice augments IL-2 and granzyme B production
CD270) was involved in the inhibition of T-cell pro-
found on circulating B cells. Furthermore, we show
+
liferation and cytokine synthesis. There is only one
that BTLA was upregulated and recruited to the BCR
T-cell effector and cytotoxic responses leading to
study on the role of BTLA in B cells, showing that it
in B cells activated in vitro. Finally, we demonstrate
the complete tumor regression. Therefore, the ca-
regulates B cell receptor signaling. Thus, the implica-
that BTLA triggering by HVEM attenuated human
pacity to reject tumor acquired by tumor-reactive
+
+
by CD4
T-cells and increases FV-specific CD4
tion of BTLA triggering for human B cells remains
B cell proliferation, upregulation of co-stimulatory
CD4 T-cells largely depends on the direct suppres-
poorly documented. B cells express germline encoded
molecules (CD80 and CD86) and production of cy-
sive activity of regulatory T-cells. We suggest that
+
Toll-like receptors (TLRs), which have emerged
tokines. Interestingly, chemokine secretion (IL-8 and
a cytotoxic CD4 T-cell immune response may be
as critical modulators of B cell effector functions,
MIP-1beta) was not affected by BTLA/HVEM ligation,
induced to enhance resistance against oncovirus-
notably in autoimmune diseases or TH1-related in-
suggesting that BTLA mediated inhibition is selective
associated tumors.
flammation. In humans, B cells are the only immune
for some but not all B cell functions. Altogether, our
population together with the plasmacytoid dendritic
data demonstrate that BTLA regulates human B cell
cells to express TLR9. TLR9 recognizes hypo-methyl-
responses, and has implications for future develop-
ated CpG motifs, characteristic of bacterial, viral and
ment of therapies modulating B cells.
+
102
103
053 | Improving Immunity
054 | Improving Immunity
Constitutive activation of the NFkB pathways in DC improves
their T-cell stimulatory capacity and IL-12p70 secretion
A concomitant interaction of CD4+ T-helper cells, DC,
and CD8+ T-cells is required for an effective boosting of tumor
­antigen-specific CD8+ T-cell expansion
1
2, 3
3
1
1,*
1
1
1
1,*
Isabell Pfeiffer , Reinhard Voll , Eva Gückel , Gerold Schuler , Niels Schaft ,
1,*
and Jan Dörrie Stefanie Böhm , Beatrice Schuler-Thurner , Gerold Schuler , Jan Dörrie , and Niels
1,*
Schaft
1
Department of Dermatology, Universitätsklinikum Erlangen, Germany
1
Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany
2
Division of Rheumatology and Clinical Immunology & Centre for Chronic Immunodeficiency,
University Medical Centre, Freiburg, Germany
*
Share senior authorship
3
Medizinische Klinik 3, Universitätsklinikum Erlangen, Germany
*
Contributed equally
+
IL-12p70, we co-cultured the transfected DC with
The necessity of CD4 T-cell help during priming
CD8 T-cells and DC. Emulation of a sequential in-
in the induction of full maturation of dendritic cells
agonistic soluble CD40 ligand (sCD40L). We added
for an effective secondary expansion of antigen-
teraction, in which DC initially interacted with the
+
T-cells was already examined in
+
sCD40L immediately or 24 h after electroporation to
specific CD8
tory capacity. Given that tumor-antigen-loaded
the DC-cultures. This did not increase the secretion
the murine system. In addition, a great influence
mature DC are a promising tool to induce signifi-
of IL-12p70, but unlike expected, the sCD40L-addi-
of CD4
cant numbers of high quality effector and memory
tion even reduced the quantity of secreted IL-12p70
lasting and robust immune response was also cor-
capacity to prime and expand the CD8
T-cells, we investigated the influence of the expres-
when sCD40L was given directly after electropora-
roborated in mice. Two distinct T-cell help models
A concomitant interaction of all three cell types,
sion of constitutively active mutants of activators of
tion. No effects were detected, when sCD40L was
have been proposed: the sequential two-cell inter-
where CD4
the NFkB-pathways, with the main aim to improve
supplemented 24 h after electroporation.
action model and the three-cell interaction model.
co-culture, still had no influence on the priming
the stimulatory capacity and their applicability in
Subsequently, we investigated the capacity of DC
However, these models were never investigated
capacity. However, upon re-stimulation (2
+
T-cell help on the generation of a long-
CD4
T-cells, which were then removed by flow
(DC) and is thus relevant for their T-cell stimula-
cytometry sorting, and subsequently encountered
+
CD8 T-cells did not significantly enhance the DC’s
+
+
rd
T-cells.
T-cells were not removed from the
nd
and
stimulation), an obviously superior antigen-­
cancer immunotherapy. Therefore, we transfected
transfected with the NFkB-pathway activators to
systematically in the human system. Thus, the de-
3
cytokine-cocktail-matured DC with RNA coding
prime and expand autologous T-cells antigen-spe-
velopment of an experimental in vitro system by
specific CD8 T-cell expansion was detected. This
for constitutively active mutants of activators of the
cifically. A clear superiority was seen upon restim-
which both models can be emulated, regarding the
improved expansion was only assessable when
classical (IKKb) as well as the alternative (IKKa)
ulation of the T-cells, while no advantage was ob-
interaction between human dendritic cells (DC),
both epitopes, the helper epitope and the CD8
NFkB-pathway. The expression of the constructs
led to up-regulation of distinct favorable surface
rd
served during priming. After the 3 stimulation, we
achieved a ten-fold higher rate of antigen-specific
+
+
+
CD4 , and CD8 T-cells, is desirable.
+
epitope, were present by the same DC. Further+
+
We equipped ex vivo generated bulk CD4 T-cells
more, antigen-specific CD8
T-cells generated by
markers (CD25, CD40, CD70, CD83, CD86, and
CD8 T-cells in comparison to the control cytokine-
with a gp100-specific TCR by RNA electroporation.
providing help in the three-cell setting maintained
OX-40L) and to an increased secretion of several
cocktail-matured DC transfected with the antigen
These cells were co-incubated with gp100-peptide-
CD27 expression, which already indicates a pre-
loaded DC to elucidate the antigen-specific cross-
memory formation upon the 1 stimulation.
+
T-cells
st
pro-inflammatory cytokines (IL-6, IL-8, TNF, and
mRNA only. The antigen-specific CD8
IL-12p70). Further, we detected that the density of
generated by stimulation with IKK-transfected DC
talk between CD4
T-cells and DC. A clear Th1
In conclusion, our human in vitro system permit-
the expressed surface markers and the quantity
also maintained CD27 expression longer, indicat-
cytokine secretion, as well as the antigen-specific
ted the comparison of both models and revealed
of secreted cytokines correlated with the quantity
ing the generation of T-cell memory. Furthermore,
up-regulation of maturation markers (CD25, CD40,
that that a simultaneous interaction of all three
of transfected RNA. However, we also observed a
the expanded T-cells were able to secrete IFNγ in
CD80, CD86, and CD70) on both immature (iDC)
cell types improved CD8 T-cell expansion when
high donor to donor variation.
response to Ag-loaded T2-A1 cells.
and mature (mDC) DC, and the up-regulation of ac-
the DC presents both epitopes. Consequently, this
+
+
+
Transfected DC produced high IL-12p70 quanti-
Recapitulatory, transfection of constitutively active
tivation markers (CD25, CD69) on CD4 T-cells was
human in vitro model can serve as a tool for further
ties. In contrast, the immunosuppressive cytokine
activators of the NFkB pathways led to an ame-
observed in a time-dependent manner. Decreasing
investigations concerning T-cell help.
IL-10 was secreted in very low quantities. This DC
lioration of the DC by enhancing critical features:
the number of helper cells resulted in a reduction of
phenotype should efficiently drive T-cells towards
(i) phenotype (up-regulation of co-stimulatory
both antigen-specific up-regulation of maturation
Th1-type immune responses. Additionally, the DC
molecules), (ii) cytokine secretion (increased and
markers and Th1 cytokine secretion.
transfected with both activators still secreted IL-
prolonged IL-12p70, with low IL-10 secretion, and
To investigate whether this antigen-specific cross-
12p70 even 48 h after electroporation.
sustained migratory capacity), and (iii) elevated
talk between DC and CD4
To investigate whether the transfected DC still have
capacity to expand autologous antigen-specific
on the priming and re-stimulation of CD8 T-cells,
the ability to respond to extracellular signals with
T-cells with a favorable phenotype.
we mimicked both the sequential two-cell and the
an increased secretion of cytokines, especially
104
+
Activation of the NFkB-pathways is a key process
+
T-cells has an effect
+
+
simultaneous three-cell interaction of CD4
and
105
055 | Improving Immunity
056 | Improving Immunity
Non-toxic application of paclitaxel reduces chronic inflammation and abrogates myeloid derived suppressor cell activity in
ret transgenic melanoma bearing mice
CD86 and IL-12p70 are key players for T helper 1 polarization
and NK cell activation by TLR-matured dendritic cells
1
1
2
1
Alexandra Sevko , Tillmann Michels , Michael Shurin , Viktor Umansky 1
German Cancer Research Center and University Hospital Mannheim, Heidelberg, Germany,
2
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
106
1
1
1
1
Bettina Otte , Felix S. Lichtenegger , Katharina Mueller , Barbara Beck , Wolfgang
1
2
1
­Hiddemann , Dolores J. Schendel , and Marion Subklewe 1
Department of Internal Medicine III, Klinikum der Universität München, Munich, Germany
2
Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center
for Environmental Health, Munich, Germany
Melanoma is one of the most rapidly growing
of p38 MAPK in MDSC by intracellular staining of
Dendritic cells (DCs) are important regulators of the
vation, especially in cancer immunotherapy. The
cancers in the world. This is a highly immuno-
phosphorylated protein on threonine 180 and tyros-
human immune response. By means of direct in-
described effects of this type of DCs particularly
genic tumor but also highly immunosuppressive.
ine 705. The intracellular accumulation of TNF-α
tercellular interactions and secretion of cytokines,
depend on their high CD86 expression and IL-12p70
Moreover, melanoma is known to be resistant to
and S100A9 was chosen as a read-out system of al-
they can induce a stimulatory or a regulatory re-
secretion.
conventional chemotherapy, which aggravates im-
terations downstream to p38. Activation of p38 was
sponse, depending on the environment in which
munosuppression. There are accumulating evi-
strongly down-regulated after paclitaxel treatment
they developed. In vitro, it is possible to imitate the
dences that immunosuppression in melanoma is
in tumor-infiltrating MDSC as well as MDSC from
development of DCs from monocytes, and the com-
driven by chronic inflammation that induces an
the bone marrow and spleen of tumor-bearing mice
position of the maturation cocktail used strongly
accumulation and activation of myeloid derived
as compared to untreated control. The number of
impacts on the function of resulting DCs.
suppressor cells (MDSC). Recently, we have dem-
tumor-infiltrating MDSC producing TNF-α was sig-
In order to increase insight into their stimulatory
onstrated that non-toxic application of paclitaxel,
nificantly decreased in paclitaxel treated tumor-
effects and the importance of the different signals,
clinically approved chemotherapeutic, stimulated
bearing mice as compared to untreated control.
we compared DCs matured by a TLR agonist-based
dendritic cell (DC) and natural killer (NK) cell ac-
As p38 is also involved in signaling pathways acti-
cocktail head-to-head with those generated from
tivity, maintained specific T-cell responses, dimin-
vated by secreted S100A8/A9 in autocrine manner,
the same healthy donors by the standard combina-
ished numbers of immature myeloid cells in wild-
we evaluated the intracellular expression of S100A9
tion of proinflammatory cytokines or by the im-
type mice and drives MDSC differentiation towards
as a representative of the S100A8/A9 complex. A
munoregulatory cytokine IL-10. We could show
DC in vitro. Furthermore, ultra-low dose paclitaxel
significant reduction in the number of tumor-in-
that TLR-induced DCs differed from other DC
induces anti-tumor immune responses in mouse
filtrating MDSC expressing S100A9 was found. No
subsets by a predominance of costimulatory rela-
transplantable tumor models without direct toxic
changes in p-STAT3 expression (a key player in
tive to coinhibitory molecules and by secretion of
effects on tumor cells.
another signaling pathway, which could regulate
high IL-12p70 levels, but no IL-10. Functionally, in
Here we used a ret transgenic mouse model of
both S100A9 levels and MDSC-mediated immune
a coculture with autologous T-cells these signals
spontaneous melanoma, which closely resembles
suppression) were found after paclitaxel treatment.
translated into an increase in activated IFN-γ se-
human melanoma regarding histopathology and
Next, we demonstrated that the ability of tumor-de-
creting Th1 cells, but not Th2 or Th17 cells, while
clinical development.
rived MDSC from paclitaxel treated animals to sup-
the proportion of regulatory T-cells was decreased.
We found that administration of paclitaxel at ultra-
press proliferation of activated T-cells is markedly
This T-cell activation and polarization was depend-
low, non-toxic doses induced a down-regulation
reduced as compared to those from untreated mice.
ent on IL-12p70 as well as CD86, but remarkably not
of various mediators of chronic inflammation like
Based on our data, we suggest that that the abro-
on CD80 signaling. Besides, TLR-induced DCs were
TGF-β, GM-CSF, IL-1β, IL-5, IL-10, IL-13, TNF-α and
gation of MDSC immunosuppressive activity and
capable of activating NK cells, this capacity being
IFN-γ in tumors from treated mice as compared
down-regulation of chronic inflammation leads to
dependent on secretion of IL-12p70 by DCs.
to the control non-treated group. Since all these
restoration of CD8 T-cell-mediated
We conclude that DCs matured with this TLR
factors are necessary for MDSC expansion and
agonist-based cocktail are highly suitable for ap-
activation, and are related p38 MAPK signaling
plication in immunotherapeutic strategies that rely
in myeloid cells, we analyzed an activation status
on a strong type 1 polarization and NK cell acti107
057 | Improving Immunity
058 | Improving Immunity
Prestimulation with 2-Hydroxy-Octadecylphosphocholine inhibits the Cytokine Secretion of Monocyte-derived Dendritic Cells
matured with Dendrophilin A201 and IFN-γ without Changing
their TH1-Polarisation
Paclitaxel potentiates endotoxin-induced maturation of human
monocyte-derived dendritic cells under serum-free condition
1
2
3
4
1
2
3
4
Bernd Hildenbrand , Frank Neumann , Dirk Lorenzen , Boris Müller-Hübenthal ,
5
6
6
1
1
Michael Huber , Marina Freudenberg , Chris Galanos , Hans-Helge Bartsch , Marc Azemar Bernd Hildenbrand , Frank Neumann , Dirk Lorenzen , Boris Müller-Hübenthal ,
5
6
6
1
1
Michael Huber , Marina Freudenberg , Chris Galanos , Hans-Helge Bartsch , Marc Azemar 1
Tumor Biology Center,Clinic for Medical Oncology, 79106 Freiburg, Germany
1
Tumor Biology Center,Clinic for Medical Oncology, 79106 Freiburg, Germany
Innaxon, Tewkesbury Business Park, GL20 8SD Tewkesbury, United Kingdom
2
Innaxon, Tewkesbury Business Park, GL20 8SD Tewkesbury, United Kingdom
Institute for Tumour Therapy, 37115 Duderstadt, Germany
3
Institute for Tumour Therapy, 37115 Duderstadt, Germany
Centre of Integrative Oncology, Paracelsus-Spital, 8805 Richterswil, Swiss
2
3
4
Centre of Integrative Oncology, Paracelsus-Spital, 8805 Richterswil, Swiss
4
5
Department of Biochemistry and Molecular Immunology, Institute of Biochemistry
and Molecular Biology, RWTH Aachen University, 52074 Aachen
5
Department of Biochemistry and Molecular Immunology, Institute of Biochemistry
and Molecular Biology, RWTH Aachen University, 52074 Aachen
6
Max-Planck-Institute of Immunobiology,79108 Freiburg, Germany
6
Max-Planck-Institute of Immunobiology,79108 Freiburg, Germany
Introduction: Lysophosphatidylcholine (LPC) is a
pre-incubation of immature MoDCs with R-OH fol-
Introduction: Paclitaxel (PTX), a clinically estab-
lipid signalling messenger molecule, which exhib-
lowed by maturation with DEN A201 ± IFN-γ led to
lished chemotherapeutic agent, does not act only
as their TH1-polarisation.
its potent pro-inflammatory activities and can effi-
twice as high an up-regulation of CD83, which was
by cytotoxicity, but also has immunomodulatory
Conclusion: The pre-incubation of human MoDCs
ciently stimulate immune cells, such as macrophag-
associated with a strong inhibition of cytokine pro-
activity [1]. While in the long run, PTX leads to a
with non-toxic concentrations of PTX led to an
es or dendritic cells (DCs) [1]. The R-configured
duction. Whereas LPC enhanced TH2-polarisation
broad immunosuppression, the intravenous appli-
exponential increase of sCD14-secretion and thus
enantiomer of 2-Hydroxy-octadecylphosphocholine
after maturation by DEN A201 even in the presence
cation of PTX can be associated with strong inflam-
enhanced the maturation of MoDCs with S-LPS ±
(R-OH) on the other hand has been shown to be a
of IFN-γ, pre-incubation of MoDCs with R-OH still
mation, especially in patients suffering from (sub-)
IFN-γ in serum-free medium. Therefore, our results
competitive inhibitor of cellular LPC-reacylation and
enhanced the TH2-polarisation when matured with
acute bacterial infection. To further elucidate this
indicate, that strong inflammation, often observed
to possess anti-tumour activity [2]. In this study we
DEN A201 alone, however, did not alter the strong
clinical phenomenon, we investigated the effect of
in patients after treatment with PTX in the presence
investigated whether R-OH could serve as a potential
TH1-polarisation seen previously after maturation
PTX on human monocyte-derived dendritic cells
of a sub-acute bacterial infection could, at least par-
inhibitor of DC-mediated inflammatory processes by
with DEN A201 in the presence of IFN-γ [3].
(MoDCs) matured with either R- or S-Lipopolysac-
tially, be explained by the increased presence of
analyzing its influence on the maturation and/or cy-
Conclusion: Pre-incubation of immature MoDCs
charide (LPS) under serum-free conditions.
sCD14.
tokine production of human monocyte-derived DCs
with R-OH at non-toxic concentrations [10-20µM]
Methods: MoDCs were generated in serum-free
(MoDCs) stimulated with the TLR4 agonist Dendro-
efficiently inhibits the cytokine secretion by MoDCs
medium supplemented with GM-CSF and IL-4 for 5
philin A201 (DEN A201) in the presence or absence
matured with DEN A201 and IFN-γ without chang-
days pre-treated with PTX (0,1-10 µM) over a period
of Interferon-gamma (IFN-γ).
ing their IL-10/IL-12p70 ratio or TH1-polarisation.
of 0 h, 24 h or 48 h and matured for 24 h with LPS
Methods: Immature human MoDCs were generated
Moreover, R-OH enhanced the up-regulation of both
(either R- or S-form) ± IFN-γ. MoDCs were phe-
in serum-free medium supplemented with GM-CSF
co-stimulatory cell surface molecules and the matu-
notypically characterised by flow cytometry and
and IL-4 for 5 days and matured for additional 24
ration marker CD83, which may potentiate anti-tu-
cytokine-profiled by ELISA for secreted IL-6, IL-10,
hours with DEN A201 ± IFN-γ. R-OH or LPC (as a
mour immune responses in vivo.
IL12p70 and soluble CD14 (sCD14).
cally characterised by FACS and cytokine-profiled
by ELISA for secreted IL-6, IL-10 and IL12p70 (biological active).
Results: Immature MoDCs showed enhanced upregulation of the co-stimulatory molecules CD80
and CD86 when incubated with LPC [10–50µM] or
R-OH [10–20µM] over a period of more than 24 hours
in serum-free medium. However, compared to LPC,
108
[1] Javeed A, Ashraf M, Riaz A, Ghafoor A, Afzal S,
Mukhtar MM. Paclitaxel and immune system. Eur J Pharm
Sci. 2009;38:283
Results: In contrast to R-form LPS, S-form (wild-
control) was added 24 hours before or simultaneously with DEN A201. Matured MoDCs were phenotypi-
IFN-γ enhanced both maturation of MoDCs as well
[1] Coutant F, Laure PC, Agougne S, Delair T, Andre P,
Lotteau V. Mature dendritic cell generation promoted by
Lysophosphatidylcholine. J. Immunol. 2002;169:1688-95
[2] Hildenbrand B, Kley JT, Haberstroh F, Freudenberg MA,
Azemar M, Unger C, Massing U. Cytotoxic Efficacy and Influence on Cellular Phospholipid Metabolism of 2-Hydroxyand 2-O-Acetyl-Octadecylphospho-cholines. Anticancer
Res. 2006;26(6):4255-62
[3] Hildenbrand B, Lorenzen D, Sauer B, Hertkorn C,
Freudenberg MA, Peters JH, Nesselhut T, Unger C, Azemar
M. IFN-y enhances T(H)1 polarisation of monocyte-derived
dendritic cells matured with clinical-grade cytokines using
serum-free conditions. Anticancer Res. 2008;28(3A):1467-76.
type) LPS did not induce the maturation of MoDCs
under serum-free conditions. Pre-incubation with
PTX or addition of sCD14 increased the sensibility
of MoDCs to S-LPS dramatically. Non-toxic concentrations of PTX induced an exponential increase
of sCD14 in the supernatant. Therefore, MoDCs
pre-treated with PTX and stimulated with S-LPS
showed phenotypic and functional characteristics
of fully matured MoDCs. Moreover, the addition of
109
059 | Improving Immunity
060 | Improving Immunity
The bacterial preparation OK432 induces IL-12p70 secretion in
human dendritic cells in a TLR3 dependent manner
Zoledronic acid-treated monocytes augment TRAIL-mediated
cytotoxicity of human NK cells
Arnt-Ove Hovden, Marie Karlsen, Roland Jonsson, Silke Appel
Andreas Lundqvist, Padraig D’Arcy, Erik Wennerberg, Dhifaf Sarhan
Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Bergen, Norway
Karolinska Institutet, Department of Oncology-Pathology, Cancer Center Karolinska,
Stockholm, Sweden
Dendritic cells (DC) used in therapeutic cancer im-
Natural killer (NK) cells are innate lymphocytes
treatment was not observed, suggesting that IFN-
munotherapy have to be able to stimulate T-cells re-
able to directly kill tumor cells through ligation of
gamma produced by monocytes is responsible for
sulting in an immune response that can efficiently
TNF-related apoptosis-inducing ligand (TRAIL) re-
the increase in TRAIL expression on NK cells. In
target the cancer cells. One of the critical hurdles
ceptors. However, peripheral blood NK cells express
vivo, a significant delayed tumor progression was
has been the lack of IL-12p70 production when
low levels of TRAIL and are unable to kill TRAIL
observed in tumor-bearing SCID/beige mice when
maturating the DC, which is rectified by using
sensitive tumors. Zoledronic acid (ZA) is a bisphos-
treated with ZA-primed NK cells compared to mice
the bacterial preparation OK432 (killed Streptococ-
phonate known to up-regulate expression of TRAIL
treated with unprimed NK cells (p=0.015). These
cus pyogenes, trade name Picibanil) to mature the
on human gamma-delta T-cells. We investigated
findings represent a novel approach to augment
cells. In order to identify the mechanism behind
whether exposure to ZA would result in similar
NK cell-mediated tumor cytotoxicity that could be
OK432 stimulation of DC, we investigated the con-
changes in human NK cells. No change in expres-
used to potentiate anti-cancer effects of adoptively
tribution of different Toll-like receptors (TLR) to
sion of TRAIL was observed on purified NK cells
infused NK cells in patients with cancer.
examine their involvement in IL-12p70 production.
when treated with ZA. However, when co-cultured
By combining different inhibitors of TLR signaling,
with monocytes, treatment with ZA resulted in a
we could demonstrate that TLR3 is responsible for
significant up-regulation of TRAIL expression on
the IL-12p70 production of DC induced by OK432.
NK cells (p=0.01). Consequently, exposure to ZA
Moreover, our data suggest that the ligand trig-
resulted in a significant increase in NK cell-mediat-
gering IL-12p70 secretion upon TLR3 stimulation
ed tumor cytotoxicity in vitro (p<0.0001). In block-
is sensitive to proteinase and partly also RNAse
ing experiments, neutralizing antibodies to TRAIL
treatment. The fact that a bacterial compound like
significantly reduced the killing of ZA-primed NK
OK432 can activate the TLR3 pathway in human
cells. Furthermore, in presence of concanamycin A,
DC is a novel finding. OK432 demonstrates a criti-
the level of cytotoxicity by ZA-primed NK cell was
cal ability to induce IL-12p70 production, which
reduced, suggesting also perforin-mediated killing
is of great relevance in DC based cancer immuno-
is enhanced upon exposure to ZA. The increase in
therapy.
TRAIL expression on NK cells was not dependent on
cell contact with monocytes since TRAIL was up-
(Hovden et al., PLoS One. 2012;7(2):e31217)
regulated in transwell assays following treatment
with ZA. When screened for cytokine production by
Luminex and ELISA, monocytes exposed to ZA had
an increased production of IFN-gamma. In assays
where neutralizing antibodies to IFN-gamma was
present, increase in TRAIL expression following ZA
110
111
061 | Improving Immunity
062 | Improving Immunity
Activation of the human immune system via Toll-like receptors
by the oncolytic parvovirus H-1 and its combination with targeted agents
Virus-specific CD8+ T-cells up-regulate PD-1 expression during
acute Friend retrovirus infection but are highly cytotoxic and
control virus replication
1
1
1
2
3
1
2
1
1
Maike Sieben , Susanne Roth , Franziska Springsguth , Christiane Dinsart , Jean Rommelaere ,
1
1
Peter R. Galle , Markus Moehler Gennadiy Zelinskyy , Lara Myers , Kirsten K. Dietze , Kathrin Gibbert ,
3
2
1
Volker Teichgräber , Kim J Hasenkrug & Ulf Dittmer 1
First Department of Internal Medicine, University Medical Center of the Johannes Gutenberg
University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
1
Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
2
Infection and Cancer Program, Department F010, and
2
3
Institut National de la Santé et de la Recherche Médicale Unité 701, German Cancer Research
Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, Hamilton,
MT 59840, USA.
3
National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany
The therapeutic use of oncolytic viruses in patients
which correlated with NFκ B translocation to the
It was reported that inhibitory molecules such as
with malignancy is a promising area of investiga-
nucleus. Using a TLR-signaling reporter plasmid
PD-1 were up-regulated on CD8
tion since they also increase the host immune re-
(pNiFty-Luc), NFκ B activity, assessed by increased
chronic immune response, and that the cells were
sponse by priming effector immune cells against
expression of an NFκ B-inducible reporter gene,
prematurely exhausted and dysfunctional in vitro.
the tumors. To date the role of Toll-like receptors
was increased following H-1PV infection. In addi-
The current study shows that most activated CD8
(TLRs) in the recognition of parvovirus H-1 (H-1PV)
tion, human DCs coincubated with H-1PV-infected
T-cells up-regulated expression of PD-1 during
and the activation of the host immune system has
SK29Mel TCLs demonstrated increased TLR3 and
acute infection and revealed a dichotomy of func-
not been characterized.
TLR9 expression. Furthermore DC co-cultures
tion between PD-1hi and PD-1lo subsets. More
Aims: We aimed to investigate the function of TLRs
with H-1PV-induced TCL combined with sunitinib
PD-1lo cells produced anti-viral cytokines such as
during oncolytic H-1PV-induced human immune
induced effective immune stimulation. Cytokine
IFNγ and TNFα, while more PD-1hi cells displayed
responses. The role of TLRs in the activation of the
levels increased after coculture of DCs stimulated
characteristics of cytotoxic effectors such as pro-
NFκ B transcription factor was characterized and
by H-1PV-infected and sunitinib treated TCLs.
duction of granzymes and surface expression of
the immunologic effects of H-1PV-induced tumor
These data suggest that H-1PV-induced TCLs stimu-
CD107a. Importantly, CD8 T-cells mediated rapid
cell lysates (TCL) on human antitumor immune re-
late human DCs at least in part through TLR-de-
in vivo cytotoxicity and were critical for control of
sponses were evaluated. Furthermore we explored
pendent signaling pathways. Thus, DC maturation
acute Friend virus replication. Thus direct ex vivo
the molecular interactions and synergistic effects
occurred through exposure to H-1PV-induced TCLs
analyses and in vivo experiments revealed high
between H-1PV, and the targeted agent sunitinib.
through TLR-signaling leading to NFκ B-dependent
CD8
Methods: Human embryonic kidney cells (HEK293)
activation of the adaptive immune system as indi-
expression during acute infection is not a marker
transfected to stably express TLRs were used to
cated by the increased expression of CD86, TLR3
of T-cell exhaustion.
further investigate the role of specific TLRs during
and TLR9. Thus H-1PV oncolytic virotherapy en-
immune activation. A human ex vivo model,
hances immune priming by different effects on DCs
HLA-A2-positive
line
and generates antitumor immunity. Furthermore,
(SK29Mel) was used to study immune responses
combined treatment with targeted agents did not
with corresponding HLA-restricted human den-
interfere with the pronounced immunomodulatory
dritic cells (DCs). Furthermore our human mela-
properties of H-1PV, but reinforced drug-induced
noma model enables to study the immune respons-
tumor cell killing. These findings potentially offer
es in the context of corresponding HLA-restricted
a new approach to tumor therapy.
human
melanoma
cell
+
T-cells during
+
+
+
T-cell functionality and indicate that PD-1
human DCs after treatment with H-1PV-induced
and sunitinib treated TCLs.
Results: In TLR-transfected HEK293 cells TLR3
and TLR9 were activated by H-1PV infection,
112
113
063 | Improving Immunity
064 | Improving Immunity
Synergistic augmentation of CD40-mediated activation of
­antigen-presenting cells by amphiphilic poly(γ-glutamic acid)
nanoparticles
Inhibition of tumor immunity by tumor-infiltrating CD4+
­regulatory T-cells in patients with primary and metastatic liver
cancer can be abrogated by soluble GITR-ligand
1
2
1
2
3
1
1
2
2
Sissela Broos , Linda C Sandin , Jenny Apel , Thomas H Tötterman , Takami Akagi ,
3
1
1
1
Mitsuru Akashi , Carl AK Borrebaeck , Peter Ellmark , Malin Lindstedt Jaap Kwekkeboom , Alexander Pedroza-Gonzalez , Cornelis Verhoef , Jan N.M. IJzermans ,
1
1
1
Maikel P. Peppelenbosch , Harry L.A. Janssen and Dave Sprengers .
1
Department of Immunotechnology, Lund University, Lund, Sweden
1
2
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
3
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan
Agonistic
anti-CD40
monoclonal
2
Department of Gastroenterology and Hepatology, and Department of Surgery,
Erasmus MC-University Medical Centre, Rotterdam, Netherlands
antibodies
The mechanisms that enable liver cancer to escape
immunity, and we propose that sGITRL may consti-
(mAbs) hold great potential for cancer immuno-
elimination by the immune system remain unclear,
tute a rational treatment for this disease.
therapy. However, systemic administration of anti-
but their elucidation may provide novel options for
CD40 mAbs can be associated with severe side-
therapeutic intervention. We investigated the influ-
effects, such as cytokine release syndrome and
ence of tumor-infiltrating regulatory T-cells (Treg)
liver damage. With the aim to increase the immu-
on tumor-specific CD4
nostimulatory potency as well as to achieve local
tients with liver cancer, using ex vivo isolated cells
drug retention of anti-CD40 mAbs, we linked an
from individuals with hepatocellular carcinoma
agonistic mAb to immune activating amphiphilic
(HCC) or liver metastases from colorectal cancer
poly(γ-glutamic acid) nanoparticles (γ-PGA NPs).
(LM-CRC). In both HCC and LM-CRC, CD4 T-cells
We demonstrate that adsorption of anti-CD40 mAb
display impaired tumor-specific responses com-
to γ-PGA NPs (anti-CD40-NPs) improved the stimu-
pared to CD4 T-cells isolated from tumor-free liver
latory capacity of the CD40 agonist, resulting in
tissue or blood. We found that CD4 CD25 Foxp3
up-regulation of co-stimulatory CD80 and CD86 on
Treg accumulate in both types of tumors, and are
antigen-presenting cells, as well as IL-12 secretion.
more potent suppressors of autologous tumor-
Interestingly, anti-CD40-NPs induced strong syner-
specific CD4 T-cell responses than Treg isolated
gistic proliferative effects in B cells, possibly result-
from blood. In LM-CRC, where Treg accumulation
ing from a higher degree of CD40 multimerization,
is most prominent, we found elevated Ki-67 ex-
enabled by display of multiple anti-CD40 mAbs
pression in tumor Treg compared to Treg isolated
on the NPs. In addition, local treatment with anti-
from tumor-free liver tissue or blood, suggesting
CD40-NPs, compared to only soluble CD40 agonist,
local proliferation of Treg at the cancer site. In both
resulted in a significant reduction in serum levels
tumors, tumor Treg show up-regulated expression
of IL-6, IL-10, IL-12 and TNF-α in a bladder cancer
of glucocorticoid-induced tumor necrosis factor re-
model. Taken together, our results suggest that an-
ceptor (GITR) compared to Treg in tumor-free liver
ti-CD40-NPs are capable of synergistically enhanc-
tissue and blood. Treatment with soluble GITR
ing the immunostimulatory effect induced by the
ligand (sGITRL) induces a decrease in the suppres-
CD40 agonist, as well as minimizing adverse side-
sion mediated by the tumor-infiltrating Treg, and
effects associated with systemic cytokine release.
restores the proliferative capacity and cytokine pro-
This concept of nanomedicine could play an im-
duction of CD4 CD25 T-cells.
portant role in localized immunotherapy of cancer.
Conclusion: Our results show that local accumu-
+
T-cell responses in pa-
+
+
+
+
+
+
+
-
lation of Treg in liver cancer restrains anti-tumor
114
115
065 | Improving Immunity
066 | Improving Immunity
The Chemotherapeutic Compound 2-Deoxy D-Glucose Prevents
Cell Surface Expression of NKG2D Ligands through Inhibition of
N-Linked Glycosylation
Direct immunomodulatory effects of zoledronic acid on
NK cells in Ewing sarcoma
1
1
Sarah Line Skovbakke , Lars Andresen , Gry Persson, Michael Hagemann-Jensen,
Karen Aagaard Hansen, Helle Jensen, and Søren Skov
Department of Biomedicin, Faculty of health and medical sciences, University of Copenhagen,
1870 Frederiksberg C, Denmark
1
S.L.S. and L.A. contributed equally to this work
116
Sarah-Kristin Müller, Christiane Chen, Bianca Altvater, Sareetha Kailayangiri, Sibylle
Pscherer, Martina Ahlmann, Sibylle Mellinghoff, Uta Dirksen, Heribert Juergens, Claudia
Rossig
University Children´s Hospital Muenster, Department of Pediatric Hematology and Oncology,
Muenster, Germany
NKG2D ligand surface expression is important
surface expression. Cancer and infection often
The aminobisphosphonate zoledronic acid (ZA)
IL-2. Activated NK cells effectively interacted with
for immune recognition of stressed and neotrans-
result in aberrant glycosylation, which could likely
can induce apoptosis of Ewing sarcoma cells
both K-562 leukemia targets and Ewing sarcoma
formed cells. In this study, we show that surface
be involved in modulation of NKG2D ligand expres-
and inhibit primary tumor growth in preclinical
cells. The sensitivity of four different Ewing
expression of MICA/B and other NKG2D ligands is
sion. Our data further imply that chemotherapeutic
models. Based on first evidence that zoledronic
sarcoma cell lines (VH-64, WE-68, TC-71, and
dependent on N-linked glycosylation.
use of 2DG may restrict NKG2D ligand surface ex-
acid combined with chemotherapy is effective in
Cado) to allogeneic NK cells was highly variable,
The chemotherapeutic compound 2-deoxy-D-glu-
pression and inhibit secretion of immunoinhibitory
refractory Ewing sarcoma, the drug has now been
but consistent among all five donors. Cytolytic de-
cose (2DG) potently inhibited surface expression
soluble NKG2D ligands.
integrated into the current EWING 2008 treatment
granulation responses by CD107a expression were
of MICA/B after histone deacetylase inhibitor
regimen where its activity is assessed as add-on
comparable in the presence and absence of increas-
treatment; the inhibition occurred posttranscrip-
treatment to adjuvant chemotherapy in a rand-
ing concentrations of ZA (0.1-10 µM) throughout
tionally without affecting MICA promoter activity.
omized manner. A promising experimental strat-
various stimulator-to-responder ratios. NK cells
Transient overexpression of MICA surface expres-
egy for the treatment of cancer is adoptive transfer
directly isolated from peripheral blood of three
sion was also inhibited by 2DG. 2DG is a known
of tumor-reactive immune effector cells. Specifi-
donors had substantially lower degranulation re-
inhibitor of glycolysis and N-linked glycosylation.
cally, Ewing sarcoma cells were remarkably sensi-
sponses (<5% CD107a+ cells among CD56+CD3-
It blocks N-linked glycosylation by a reversible
tive to targeting by activated NK cells. Combina-
NK cells) to both K-562 and Ewing sarcoma targets,
mechanism that can be alleviated by addition of D-
tion strategies of novel drugs and cellular targeting
and again, responses were unaffected by the pres-
mannose; this does not, however, affect the inhibi-
have only recently started to be explored. Besides
ence of ZA. We conclude that ZA does not interfere
tion of glycolysis. Addition of D-mannose restored
their antitumor and antiresorptive effects, amino-
with cytolytic NK cell responses to Ewing sarcoma
MICA/B surface expression after 2DG treatment.
bisphosphonates have immunomodulating effects
targets. Ongoing experiments address the immune
In addition, specific small interfering RNA-medi-
that may contribute to or interfere with their antitu-
phenotype and activating receptor expression of
ated targeting of glycolytic enzymes did not affect
mor activity. Well-described effects include activa-
NK cells expanded in the presence and absence of
MICA/B surface expression, strongly suggesting
tion of γδ T-cells and indirect activation of NK cells
ZA. Moreover, NK cell phenotype and function are
that N-linked glycosylation, and not glycolysis, is
via DC-like cells. Here we investigated whether ZA
investigated ex vivo in Ewing sarcoma patients un-
essential for MICA/B surface expression. NK cell-
directly affects activation and functionality of NK
dergoing treatment with ZA compared to matched
mediated killing assay and staining with a recom-
cells, and whether the drug interferes with cytol-
controls.
binant NKG2D–Fc fusion protein showed that all
ytic responses of NK cells to Ewing sarcoma cells.
Our current approach and further studies aimed to
functional NKG2D ligands induced by histone dea-
NK cell lines were generated from five healthy
determine the interactions of novel drugs, targeted
cetylase inhibitor treatment were abolished by 2DG
donors and two patients with Ewing sarcoma by
therapies and immunotherapy may lead to more ef-
treatment and fully reconstituted by further addi-
a single round of in vitro stimulation of peripheral
fective combination strategies.
tion of D-mannose.
blood mononuclear cells with K562mb15CD137L
Our data suggest that posttranslational N-linked
stimulator cells and subsequent 10-day expansion
glycosylation is strictly required for NKG2D ligand
in the presence of 40 IU/ml recombinant human
117
067 | Improving Immunity
068 | Improving Immunity
Enhancement of cellular immune response against tumor cells
using PAMPs
Optimized human Granzyme B based immunotoxin to circumvent the inhibitory potential of SerpinB9 during cancer therapy
1
1
1
2
1
Claudia Maletzki , Ulrike Klier , Saskia Stier , Ernst Klar , Michael Linnebacher 1
Section of Molecular Oncology and Immunotherapy
²
Department of General Surgery, University of Rostock, Germany
118
2
3
1
3
Sonja Schiffer, Grit Hehmann-Titt , Theo Thepen , Michael Huhn , Rainer Fischer ,
1, 3
Stefan Barth 1
Dept. of Experimental Medicine and Immunotherapy, RWTH Aachen, Helmholtz Institute for
Biomedical Engineering, Pauwelsstr. 20, 52074 Aachen, Germany
2
Pharmedartis GmbH, Forckenbeckstrasse 6, 52074 Aachen, Germany
3
Dept. of Pharmaceutical Product Development, Fraunhofer IME, Forckenbeckstr. 6, 52074
Aachen, Germany
This study addresses the question of whether
strong effects observed in vitro, Taxol and Resiqui-
The human origin and its efficient ability to kill
shown before (Stahnke et al., 2008) However, the
pathogen-associated molecular pattern (PAMP-)
mod mediated substantial tumor growth control,
even resistant tumor cells puts the potential of
apoptosis inducing effect of Gb was significantly
substances are applicable as active immunothera-
leading to >50 % reduction of tumor volumes. Of
Granzyme B (Gb) as part of a “magic bullet” in
decreased in PI9 positive targeT-cells. Here, the im-
peutic compounds for colorectal cancer (CRC) in
note, this was even better than the standard CRC
cancer therapy more and more into focus. One of
munotoxin Gb-Ki4 was determined. Ki4 is a single
vitro and in vivo.
chemotherapeutic drug Irinotecan. Raised levels of
the major drawbacks of Gb in tumor therapy is the
chain variable fragment (scFv) targeting CD30, an
Primary human CRC cell lines (HROC40, HROC60,
activated CD166+ leukocytes were involved in the
expression of Serpin B9 (PI9), the main inhibitor of
internalizing tumor antigen highly overexpressed
HROC69) were treated with increasing concentra-
PAMP-mediated antitumor response.
Gb, within many tumor cells. Therefore it is nec-
on Hodgkin lymphoma cells. In our group S. Barth
tions (0.01 – 10µM) of TLR-agonists (Taxol, Re-
Data presented herein prove the therapeutic po-
essary to find a variant of Gb which can induce
and M. Huhn (Blood 2000) already demonstrated
siquimod, LPS, Poly I:C, and CpG ODN) for 24,
tential of PAMPs, resulting in both tumor growth
apoptosis independently of PI9.
the highly potent activity of the first recombinant
48, 72 hours and 7 days. As determined by flow
inhibition and potent immune activation. These
SerpinB9 belongs to the large superfamily of serine
anti-CD30 immunotoxin Ki4-ETA isolated from
cytometry, treatment with Taxol resulted in a
findings make them very promising candidates for
protease inhibitors which regulate several kinds of
E. Coli against disseminated Hodgkin lymphoma
dose and time-dependent growth inhibition in all
further optimization of immune-based therapies.
proteases and are involved in processes such as
in SCID mice.
cell lines (>70 % vs. control). This effect was ac-
These include applications as single or combina-
tumorigenesis, apoptosis and inflammation. The
We evaluated the expression of PI9 in different
companied by reduced cell viability (~ 20 % vs.
tory agents for active unspecific therapies, adjuvant
42 kDa protein is the main inhibitor of Granzyme
CD30 positive cancer cell lines on protein level.
control). However, tumor cells were only margin-
standard regimens or even as adjuvants in cell-
B mainly expressed within cytotoxic T lympho-
Prior to cytotoxicity testing SNAP tag technology
ally influenced by the remaining PAMPs (Resiqui-
based immunotherapies.
cytes and natural killer cells but also in endothe-
was used to demonstrate the cellular uptake of Ki4.
mod > LPS > Poly I:C > CpG ODN) even after 7
lial or epithelial cells. It can protecT-cells against
Furthermore we verified that the presence of PI9 in
days. The immunostimulatory capacity of PAMPs
self-inflicted injury or misdirected Granzyme B
CD30 positive targeT-cell indeed prevents apoptosis
to mediate antitumoral responses was then ex-
(Bots et al., 2006). The pro-apoptotic serine pro-
by complexing Gb whereas treatment with Gb-Ki4
plored in co-culture experiments (48h, 72h) using
tease Gb is stored in the cytotoxic granules of the
led to apoptosis in cells not expressing PI9. The
peripheral blood leukocytes from healthy volun-
above mentioned effector cells of the innate and
main objective of this study was therefore the de-
teers. Numbers of residual tumor cells were taken
adaptive immune system. It is the main effector
velopment of Serpin B9 independent Gb variants.
as measure of antitumoral effects. In these co-
molecule involved in host rejection of tumor and
Using computational approaches we identified po-
culture experiments, antitumoral effects were dra-
virally infected cells. During the last few years,
tential mutants which remain their enzymatic po-
matically boosted. Most pronounced effects were
many tumor cells have been described to express
tential but at the same time are not inhibited by PI9
observed for Taxol (10 µM, 72h) and Resiquimod
SerpinB9 in order to escape immune surveillance
anymore. Seven different mutants were generated
(all concentrations, 72h), with up to 95 % tumor
by tumor-infiltrating CTL or NK cells. Promising
by site-directed mutagenesis and could successful-
cell killing. In a subsequent series of in vivo ex-
approaches for targeted tumor therapy, however,
ly be expressed secretory in HEK293T-cells with the
periments, CT26-tumor carrying Balb/c mice were
are Gb based immunotoxins (composed of a cell
same yield and stability as the wildtype enzyme.
systemically treated with PAMPs (6 injections in
specific moiety linked to a cytotoxic part) because
All mutants were tested in vitro for their activity in
total, twice a week, n=5 per group). Control mice
of their human origin not provoking undesirable
presence or absence of recombinant PI9. Thus, this
received saline (n=5). Tumor growth was moni-
immune responses. In our group the potential of a
work highly contributes to the optimization of Gb
tored for 28 days, lymphocyte subpopulations
Gb based immunotoxin directed against CD64-pos-
based immunotoxins and improves its possibility
were examined from blood samples. Similar to the
itive malignancies like AML has successfully been
to become clinically relevant in the near future.
119
069 | Improving Immunity
070 | Improving Immunity
CTLA-4 antibodies Tremelimumab and Ipilimumab
overcome tumor-mediated immunsuppressive effects on
human dendritic cells
Targeting of Macrophage Galactose-type C-type Lectin (MGL)
induces DC signaling and activation
1
1
1
1
1
K. Göpfert , P. Schäfer , M. Sieben , H. Jonuleit , H. Dally³, F. Hermann², P. R. Galle ,
1
M. Moehler 1
University Medical Center of the Johannes Gutenberg University Mainz, Langenbeck-Street 1,
Mainz
2
Bristol-Myers Squibb, München, Arnulfstr. 29
3
Pfizer Pharma GmbH, Berlin, Linkstr. 10
1
1
1
1
Department of Experimental Medicine, “Sapienza” University, Rome, Italy
2
Center for Glycomics, Department of Cellular and Molecular Medicine and School of Dentistry, University of Copenhagen, Denmark
3
Department of Molecular Medicine “Sapienza” University, Rome, Italy
Tumors have distinct mechanisms to circumvent
place four hours prior coculture of Tregs with iDCs
Dendritic Cells (DCs) are the most potent antigen
homo-dimers on DC plasma membrane, promotes
the human immune system. Expression of CTLA-4
by adding 2µg/ml anti-CD3 and 2µg/ml anti-CD28.
presenting cells and are employed in cancer vacci-
the phosphorylation of Erk 1/2 MAP kinases and
(cytotoxic T-lymphocyte-associated antigen 4) on
The human Sk29Mel melanoma cells clearly ex-
nation. Several receptors are being studied in order
triggers NFkB classical pathway. The activation of
tumors and regulatory T-cells (Tregs) can lead to
pressed CTLA-4 on the surface, measured by extra-
to identify strategies that combine the cross-presen-
intracellular signals leads to DC phenotypic matu-
suppression of immune defence mechanisms. So
and intracellular FACScan analyses. In coculture,
tation of the antigen to optimal DC activation. The
ration, with a concomitant reduction of phagocyto-
far CTLA-4-mediated blockage of human dendritic
the oncolytic parvovirus H1-infected (H-1PV)
C-type lectin Macrophage Galactose type C-type
sis and an enhanced migration capacity (25-30%).
cells (DCs) by CTLA-4 expressing tumors have not
SK29Mel cell lysates induced maturation of iDCs.
Lectin (MGL), expressed by DCs, is a receptor in-
After MGL activation, DCs induce a strong prolif-
been analysed in vitro or ex vivo models. As CTLA-4
Using ELISA for determining cytokine release we
volved in the recognition of GalNAc (Tn)-carrying
eration of allogeneic T-cells and stimulate a strong
receptors can be blocked efficiently by new CTLA-4
could also show an increase in TNF-α release but
antigens. We previously demonstrated that the
CD8 and CD4 T-cell-mediated IFNgamma produc-
antibodies tremelimumab or ipilimumab, we ana-
we are unable to show strengthening by adding
tumour-associated antigen (TAA) Tn-MUC1 inter-
tion. These results demonstrate that MGL engage-
lysed these antibodies in our ex vivo human mela-
tremelimumab or ipilimumab. The further adding
nalized through MGL was cross-processed by DCs
ment profoundly affects DC plasticity inducing and
noma model for their effects on maturation of DCs
of cytototoxic T lymphocytes (CTL) showed also an
when administered as glycopeptide (MUC19Tn, 60
directing a Th1 immune response. Moreover, MGL
and their role on Tregs.
H-1PV addicted increase in IFNγ and no accessorily
mers), while remained blocked in HLA class II com-
receptor expressed on human DC can be targeted
Tregs were isolated from human Buffy coats from
effect of ipilimumab and tremelimumab.
partment when used as glycoprotein (320 mers). In
by glycopeptide based vaccines with adjuvant ac-
HLA-A2 restricted donors with magnetic beads
In our second part we tried to overcome the nega-
this study, we investigated the possibility of stimu-
tivity and tumour specificity.
and monocytes were isolated using adherence. For
tive effect of Tregs on DC maturation. Here, we
lating MGL receptor to increase DC performance by
differentiation of monocytes into immatured DCs
found an increase in IL-6 and decrease in TGF-β in
using the MUC19Tn as a model of Tn-TAA and the
(iDCs), monocytes were stimulated with IL-4 and
the supernatants after coincubation with tremeli-
anti-MGL antibody (MLD-1) as a classical receptor
GM-CSF and maturation of iDCs was induced by
mumab or ipilimumab. Icreased IL-6 and decreased
binder.
a cytokine cocktail (CC), containing 0,01µg/ml
TG-β correlates with a pro-inflammatory milieu and
DCs were derived from PBMCs of 6 healthy donors
TNFα,IL-6, IL-1β and 1µg/ml PGE2. The Parvovi-
stronger T-cell activation and proliferation. The in-
and were analyzed before and after MGL engage-
rus induced tumor cell lysis was used as model to
hibiting effect of Tregs on matured DCs cannot be
ment using MUC19Tn and MLD-1, as stimulators.
show the induction of specific antiumor immune
abrogated by tremelimumab and ipilimumab.
DC phenotype, endocytosis, migration and IL-10/
responses (Moehler et al. HGT 2005). Parvovirus
To our knowledge this is the first direct analysis that
IL-12 secretion were evaluated by cytofluorimetry.
H1-infected and uninfected Sk29Mel melanoma
tremelimumab and ipilimumab can partially over-
Allo T-cell-stimulating capacity and IFNgamma
cells were cocultured with iDCs with or without
come the negative feedback of Tregs on human DCs.
T-cell production were estimated by H-thymidine
tremelimumab (10µg/ml) and with or without ip-
These results clarify the importance of CTLA-4 as
uptake and ELISpot assay, respectively. DC intra-
ilimumab (10µg/ml) in a ratio of 1:3 for 3 days.
therapeutical goal for treatment of human tumours
cellular signaling and MGL oligomerization were
Tregs were cultured in RPMI saturated with 10%
to overcome tumor-induced immune suppression.
studied by Western Blot and confocal microsco-
human plasma and 50 IU/ml IL-2. Activation took
120
1
Chiara Napoletano , Ilaria Grazia Zizzari , Aurelia Rughetti , Hassan Rahimi ,
1
2
2
3
1
Valeria Visconti , Henrik Clausen , Hans H. Wandall , Francesca Belleudi , Filippo Bellati ,
1
1
Luigi Frati , Marianna Nuti 3
py. MGL engagement induces homo-trimers and
121
071 | Improving Immunity
072 | Improving Immunity
Exploring the feasibility of future combinatorial approaches of
chemotherapy with immunotherapy for melanoma – influence
of chemotherapeutic drugs on the functions of immune cells
Updated results from a phase 2-3 clinical study in patients with
advanced colorectal carcincoma treated with the immunomodulator MGN1703 – the IMPACT study
Stefanie Gross, Anna Vogel, Waltraud Leisgang, Annett Hamann, Carmen Trütschel,
Gerold Schuler, Eckhart Kämpgen
M. Tschaika , H. J. Schmoll , J. Riera-Knorrenschild , F. Mayer , J. Kuhlmann ,
6
7
1
1
1
1
8
J. Trojan , ­V. I. Vladimirov , E. Weith , M. Schroff , M. Krikov , M. Schmidt , B. Wittig for the ­IMPACT Study Team
1
2
3
4
5
Department of Dermatology, University Hospital Erlangen, Germany
1
Mologen AG, Berlin, Germany
2
University Clinic Halle (Saale), Halle, Germany
3
Universitätsklinikum Giessen und Marburg, Marburg, Germany
4
Medical Clinic, University of Tübingen, Germany
5
Universitätsklinikum Freiburg, Freiburg, Germany
6
Johann Wolfgang Goethe University, Frankfurt, Germany
7
State Budget Medical Institution Stavropol, Patigorsk; Russia
8
Foundation Institute Molecular Biology and Bioinformatics, Freie Universität Berlin, Germany
Classical cytotoxic anticancer agents and also the
ability and T-cell effector funtions in most cases.
Introduction: This phase 2-3 study is performed in
Results and discussions: The patients received
new class of more specific kinase inhibitors are
However, differential kinetics and effects on pre-
patients with advanced CRC having disease control
up to 132 treatment administrations, so far. The
able to shrink large tumor masses in only a short
existent memory T-cell responses, priming of naive
after first-line therapy. The patients are treated
majority of adverse events (84%) reported in the
time in many patients, however often success is not
T-cells or release of suppressed T-cells by therapeu-
with the synthetic DNA-based immunomodula-
study have been assessed as not drug-related by
durable. Immunotherapy has shown long-time re-
tic antibodies show the requirement of further fine-
tor MGN1703 which acts as an agonist of toll-like
the investigator. The remaining AEs (16%) include
sponses though time to show those effects is long.
tuning of treatment schedules.
receptor 9. The objective of the study is to assess
mild fever, injection site itching, muscle aching, ar-
Neither of these two types of treatment by itself
efficacy and safety of the MGN1703 treatment in
thralgia, fatigue, paresthesia, rash, and moderate
has been sufficient to cure cancer, but, combin-
comparison to placebo.
subfebril temperature and increased ANA in single
ing chemotherapy with immunotherapy may allow
Material and methods: The IMPACT study is de-
patients. Currently, nine serious adverse events
improving therapeutic results and expanding their
signed as a randomized double-blind placebo-
(SAEs) have been reported of which only one was
durability.
controlled phase 2-3 study, in which patients with
assessed as probably drug-related (“atypical pneu-
The effects of different chemotherapeutic agents
advanced CRC showing disease control after first-
monia”). Local reactions include such symptoms as
on immune cells were studied at different time-
line therapy with standard chemotherapy regimen
mild redness and swelling at injection site reported
points ex vivo in melanoma patients curently under
are included. The study treatment (60 mg MGN1703
in single patients. No laboratory or clinical signs of
chemotherapeutic treatment and in cells of healthy
or placebo) is administered subcutaneously twice
dose-limiting toxicities have been reported, so far.
donors after in vitro preincubation. The peripheral
weekly in a ratio 2:1, respectively. The therapy is
Conclusions: The preliminary safety results of the
blood mononuclear cells (PBMC) were exposed to
continued until tumor progression, intolerable tox-
ongoing clinical study in patients with advanced
different chemotherapeutic agents then the cell vi-
icity, exclusion criteria or withdrawal of consent.
CRC show that treatment with MGN1703 at the
ability and phenotype were analyzed by flow cy-
The study is conducted in Germany, Austria,
dosage of 60 mg is well tolerated and safe. Reported
tometry. Effects of the drugs on antigen-specific
France, Czech Republic and Russia. One hundred
AEs which were assessed as possibly drug-related
T-cell function were analyzed by specific cytokine
twenty nine patients are planned to be included
belong to expected study drug reactions known for
release and proliferation using two-color fluoros-
into the study. The efficacy and safety of the study
immune modulating drugs.
pot (fluorescent ELIspot), multiplex cytokine-bead
treatment will be evaluated based on extensive im-
array and multifunctional T-cell assay (MFTC).
munological tests, radiological assessment, safety
At concentrations up to two fold of the plasma level
laboratory results and assessments of the quality
found in patiens, most chemotherapeutic agents
of life.
showed only marginal effects on lymphocyte vi122
123
073 | Improving Immunity
074 | Improving Immunity
Effective chemo/immunotherapy in melanoma patients
­activates non-canonical AKT pathway of Melan-A+
­tumor-­specific CD8+ T-cell clones
New ways to improve the treatment of rectal cancer
patients with liver metastases using metronomic regimen
of chemo­therapy with recombinant inferferon-alpha
1
1
2
1
1
1
1
2
2
1
C. Di Donna , B. Palermo , O. Franzese , D. Del Bello , N. Gualtieri , C. Nuzzo ,
3
1
4
1
E. Proietti , V. Ferraresi , C. Catricalà , P. Nistico .
Yuri Kudryavets , Grigory Maksim`yak , Victor Zhylchuk , Ada Vorontsova ,
1
1
Natalya Bezdenezhnykh , Vasilij Chekhun 1
Regina Elena National Cancer Institute, Rome, Italy,
1
2
Department of Neurosciences, University of Tor Vergata, Rome, Italy,
RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Kyiv, Ukraine
3
Istituto Superiore di Sanità, Rome, Italy,
2
Rivne Region Hospital, Rivne, Ukraine
4
Department of Dermatology-Oncology, S. Gallicano Dermatological Institute, Rome, Italy
2
bined treatment, a non-canonical AKT activation
Traditionally for the therapy rectal cancer (RC) pa-
can (2.0 mg/m ) + cisplatin (2.5 mg/m ) twice a
the effectiveness of the antitumor immune response
was observed. The identification of the extracel-
tients use chemotherapeutic (CT) schemes based on
week, group IV (58 patients) – irinotecan (2.0 mg / m )
2
2
associated with clinical efficacy is an attractive an-
lular stimuli and signaling pathway responsible
various combinations of oxaliplatin, irinotecan, fluo-
+ cisplatin (2.5 mg/m ) twice a week combined
titumor strategy, although molecular and cellular
for DTIC-mediated activation of the AKT signaling,
ropirymidines and methods of regional CT. However,
with daily administration i/m IFN-alpha at a dose
mechanisms by which conventional chemothera-
are currently under investigation. A phase II rand-
in most cases tumors remain resistant to CT and
1x10 IU; Group V (26 patients) – cyclophosphamide
peutics can activate the immune system against
omized clinical trial with DTIC administered one
therefore requires finding new ways to palliative
(15 mg/m , 2 g / week per os); group VI (21 patients)
cancer are still under investigation. In a pilot clini-
day before peptide-vaccination (Melan-A and NY-
treatment of patients with metastatic rectal cancer.
– cyclophosphamide (15 mg/m , 2 g / week per os)
cal trial, in clinically disease free HLA-A2 mela-
ESO-1) plus IFN-α, versus peptide-vaccination plus
In recent years has explored new modes of chemo-
+ IFN 1 x10 IU daily, i/m. The duration of therapy
noma patients, we have reported that the combi-
IFN-α alone, is ongoing in our Institution, to prove
therapy, in particular “dose-dense”, “chemo-switch”
was 12-15 months, the results were assessed by 24-30
nation therapy using the chemotherapeutic agent
the clinical efficacy in the prevention of melanoma
and “metronomic” modes. The last one, as it seems,
months.
dacarbazine (DTIC) one day before peptides vac-
relapse in clinically disease-free HLA-A2 patients
6
2
2
6
has as a target the tumor neoangiogenesis and espe-
Analysis of the clinical effectiveness of metronomic
cination (Melan-A and gp100) plus IFN-α increased
cially effective in combination chemotherapy with
mode drug therapy in patients with mCRC demon-
the number of tumor-reactive long-lasting effector-
antiangiogenic drugs.
strated high performance such mode chemotherapy
memory CD8 lymphocytes. To identify the mecha-
Among the wide spectrum of angiogenesis inhibi-
compared with conventional treatment using irinote-
nisms enhancing the immune response, induced
tors special attention is given interferon (IFN) which
can. Among the most effective chemotherapeutic
by DTIC combined with peptide-vaccination, we
is well known as a natural non-toxic drug with an-
schemes for all parameters (p <0,001) appeared met-
analyzed the endogenous and treatment-induced
tiangiogenic multitarget action that prevents blood
ronomic mode application irinotecan with cisplatin.
antigen specific CD8 T-cell response in a panel of
+
capillaries formation in tumors. Unlike other antian-
The duration of partial clinical effect in this scheme
clones isolated from six patients. We ana-
giogenic drugs IFN can simultaneously affect differ-
grew almost three times against this by using only a
lyzed the sequence of the TCR β-chain of these
ent parts of the regulation of angiogenesis. The main
high dose of irinotecan, and duration of the stabiliza-
clones and the molecular results were correlated
advantage of this cytokine is its ability to specifically
tion process and the overall survival rate increased
with the expression of CD27/CD28 co-stimulatory
inhibit tumor neovascularization without violating
respectively by 81% and 34.5%. Metronomic therapy
molecule, AKT activation and anti-tumor lytic ac-
the physiological state of angiogenesis in the body.
with cyclophosphamide was less effective, but in
tivity. The combination of chemo/immunotherapy
The paper presented the experience of treatment pa-
combination with IFN was not inferior to the effi-
elicited in Melan-A-specific, but not in gp100 clones,
tients (stages T1-4N0-2M1) with metronomic regime
ciency of irinotecan at a dose of 85 mg/m adopted.
a renewal of high-avidity/tumor-reactive T-cell
of chemotherapy. In this mode of treatment 168 pa-
Using of IFN in all the metronomic chemotherapy
clones, with a broadening TCR diversity in long-
tients carried out using different schemes, which in-
schemes significantly increased their effectiveness.
surviving patients, suggesting that the selection of
cluded cyclophosphamide, cisplatin and irinotecan
Importantly, we observed decrease of the level of
immune-resistant tumor variants may be circum-
in combination these drugs with IFN.
VEGF in the patients sera from 430-470 pg/ml to
vented by this combination therapy. In the gp100
Studied group of patients included: Group I (control,
230-250 pg/ml after 4 month therapy with IFN. So
clones, AKT activation (pSer473-AKT) canonically
44 patients) – received symptomatic treatment. Pa-
accordingly our data metronomic chemotherapy of
correlates with CD28 and/or CD27 expression, and
tients II group (54 patients) received treatment with
patients with metastatic rectal cancer is a new and
+
CD8
124
2
Combination of chemo-immunotherapy to increase
2
2
this was independent of the treatment. Differently,
irinotecan in 85 mg/m adopted the accepted proto-
perspective approach that provides inhibition of
in the late-differentiated CD27/CD28-negative tu-
col; III-VI main groups of patients receiving drug in
the disease progression, possible by antiangiogenic
mor-lytic Melan-A clones, isolated after the com-
metronomic mode: Group III (63 patients) – irinote-
mechanisms of action.
125
075 | Improving Immunity
076 | Improving Immunity
Combining Gemcitabine and RIG-I signaling
for ­chemo­immunetherapy of pancreatic cancer
Immune-complexes formed by intracellular tumor antigens
­released by chemotherapy and an injected antigen-specific
­antibody have an adjuvant effect to activate the adoptive
­immune system against cancer
Hélène Bourhis, Sabine Hoves, Peter Düwell, Mareike Stieg, Sarah Buhlert,
Jonathan E
­ llermeier, Max Schnurr
Takuro Noguchi , Stylianous Bournazos , Sacha Gnjatic , Hiroshi Shiku ,
5
2
1
Hiroyoshi Nishikawa , Jeffery Ravetch , Gerd Ritter Division of Clinical Pharmacology, Ziemssenstraße 1, 80333 Munich, Germany
1
Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer Center,
New York, U.S.A
2
Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, U.S.A
3
Department of Cancer Vaccine, Mie University Graduate School of Medicine, Mie, Japan
4
Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan
5
Experimental Immunology, Immunology Frontier Research Center, Osaka University, Osaka, Japan
1
2
1
3, 4
Pancreatic cancer is the 4th leading cause of can-
Monoclonal antibody (mAb) therapy against tumor
area of extravasations was correlated with the dose
cer-related death and the prognosis is very poor
antigens expressed on the cell surface of tumors
of antigen and the dose of antibody injected into
with a 5-year survival rate less than 5%. It is char-
such as Her2/neu and CD20 has become a standard
the mice. In addition, we found that the amount
acterized by a large immune suppressive network,
treatment for cancer. The antibodies are thought to
of spontaneous endogenous tumor antigen-specific
early metastasis and irradiation. Although chemo-
typically work through mechanisms such as direct
antibody in tumor bearing mice was not enough to
therapy with Gemcitabine is the gold standard it
interruption of the cell surface receptor engaged in
elicit the acute inflammation. This suggests the im-
only has little impact on the patients survival.
the tumor cell proliferation, and immune activation
portance of maintaining high titer of intracellular
Retinoic acid inducible gene I (RIG-I) is a ubiqui-
through Fc receptors. Recently we have established
antigen targeting antibody and of accentuating the
tously expressed cytosolic helicase detecting viral
a murine tumor model for a combination therapy
release of antigen from tumor cells to expose more
RNA by its 5‘-triphosphate moiety. 5’-Triphosphat-
of a chemotherapeutic drug and an antibody tar-
antigens e.g. by chemotherapy. Activation of innate
modified RNA was synthesized per in-vitro tran-
geting an intracellular tumor antigen (Noguchi T,
immunity by ICs may contribute to the adjuvant
scription and activation of RIG-I leads to a type-I IFN
et al. Cancer Res. 2012). Chemotherapy accentu-
effect of ICs to mature DCs in the model.
response, upregulation of MHC class-I molecules
ated the release of intracellular antigen from dying
and secretion of proinflammatory cytokines. Fur-
tumor cells to form immune-complexes (ICs) with
thermore, it induces potent apoptosis in tumor cells.
an injected antigen-specific antibody, and result-
Here we show that suboptimal doses of Gemcit-
ing in the augmentation of tumor growth inhibition
abine trigger an upregulation of the pro-apoptotic
when compared to chemotherapy alone. Although
molecules Puma and Noxa. Combination of both,
tumors without chemotherapy also released spon-
Gemcitabine and RIG-I signaling leads to higher
taneously tumor antigens from dying cells (usually
sensitivity of the tumor cells to apoptosis in a syn-
later than those with chemotherapy), ICs forma-
ergistic manner. Moreover, dendritic cells were
tion in this context was not associated with tumor
able to phagocytose killed tumor cells and upregu-
growth inhibition. We reported on a critical role
late costimulatory molecules on their surface after
of the DCs-CD8 T-cells cross-presentation axis in
co-culture with the tumor cells.
our model, but the involvement of other Fc recep-
The data presume that combination of the chemo-
tors bearing immune cells remained to be investi-
therapeutic Gemcitabine with 5’-Triphosphat-RNA-
gated. In order to study the involvement of innate
mediated RIG-I signaling has a synergistic effect on
immunity in our model, we used a reverse Arthus
tumor cell death. This tumor cell death is immuno-
passive reaction assay to visualize acute inflamma-
genic and may represent an innovative therapeutic
tion induced by ICs by determining the extent of ex-
approach of pancreatic cancer therapy.
travasation. Acute immune reaction was elicited in
+
an antigen-specific and Fc dependent manner. The
126
127
077 | Improving Immunity
078 | Improving Immunity
The Role Of Combining Synthetic Imidazoquinoline Toll-like
Receptor (TLR) Agonists And GM-CSF Activity For Potentiating
Cell Activation In Autologous Cellular Immunotherapy (ACI)
Sharply discordant biological properties of synthetic noncoding
dsRNA of different size: translational opportunities in cancer
1
1
1
1
1
1
Craig Meagher , Jason Chinn , Felecia Wagener , Crystal Cummings , Shaarwari Sridhar ,
1
1
1
1
2
Chris Ramsborg , Kien Khuu-Duong , Xinhui Ge , Lisa Martel , Mark Tomai , and James
1
Trager 1
Dendreon Corporation, Seattle, WA 98101, USA
2
3M Drug Delivery Systems, St. Paul, MN 55144, USA
1
Department of Research and Development, MultiCell Technologies, 68 Cumberland St., suite
301, Woonsocket, RI, 02895, USA
2
Division of Cellular and Molecular Biology, Toronto General Research Institute, University
Health Network, Toronto, Ontario, M5G 2C4, Canada
antigen uptake and processing and TLR7/8 stimula-
Liver cancer still represents a significant unmet
ment of asymptomatic or minimally symptomatic
tion matures APC in the ACI setting.
medical need. Noncoding dsRNA have been shown
metastatic castration resistant prostate cancer, is the
Next, the potential for TLR agonists to activate T-cells,
to stimulate various signal transduction pathways
first FDA-approved ACI. Here we describe a series
as determined by cell surface expression of activa-
through TLR, MDA6 and RIG-I-mediated recogni-
of studies for the development of an ACI targeting
tion markers (CD25, CD54, CD137, CD278), was in-
tion, leading to cellular and immunological out-
carbonic anhydrase IX (CA9) as a potential therapy
vestigated. Of the agonists tested, simultaneous
comes. We analyzed systematically the biologi-
for cancers including renal, lung, colon, and cervi-
stimulation of both TLR7 and TLR8 with R848 was
cal activity of synthetic dsRNA and analogues of
cal cancer. Like Provenge®, this new ACI utilizes
the strongest at potentiating T-cell activation. Rela-
defined size and chemical composition, on cancer-
a recombinant antigen consisting of CA9 linked to
tive to cultures supplemented with only CA9:GM-CSF,
ous and other types of cells. There was a strikingly
GM-CSF (CA9:GM-CSF), and in these studies, the
R848 enhanced frequency of activated T-cells across
different effect of such dsRNAs on cells: while short
+
+
effects of incorporating proprietary imidazoquino-
several subsets (CD4 , CD8 , γ/δ). R848 enhanced
(5bps) polyA:U showed an anti-proliferative / cyto-
line agonists that selectively stimulate TLR7 and 8
the frequency of cells expressing CD25 and CD54 ex-
toxic effect, longer sized (70bps) polyA:U showed
+
on in vitro measures of ACI potency are investigated.
pression for each subset, CD137 on only CD8 T-cells,
Initially, we compared the phenotype of large APC
and CD278 on only CD4
T-cells. In comparison,
consistent with the model that dsRNAs of differ-
following culture of PBMC from normal healthy
stimulation via TLR8 only enhanced the frequency
ent size are primarily engaged by distinct recep-
donors with either CA9:GM-CSF or CA9:GM-CSF plus
of γ/δ T-cells expressing CD278, while surprisingly,
tors and recognition pathways, a reflection of their
TLR7/8 agonists. While antigen derived GM-CSF ac-
with respect to the markers characterized, selec-
pleiotropism and the importance of rigorous RNA
tivity strongly activated APC, characterized by en-
tive stimulation through TLR7 did not significantly
monitoring in general. Based on these findings
hanced cell surface expression of CD40, CD54, CD80
enhance cellular activation of any subset. In associa-
and advances in RNA manufacturing, we are con-
and CD86, each TLR7/8 agonist was sufficient to
tion with the observed pattern of T-cell activation, a
ducting a comprehensive preclinical evaluation of
further enhance CD80 expression. Differential regu-
marked increase in the accumulation of proinflam-
synthetic short dsRNA (MCT 485) and longer sized
lation of CD86 was observed in response to TLR7 and
matory Type-1 like associated growth factors (IFN-γ,
dsRNA (MCT 465) of precise size and chemistry,
TLR8 stimulation that enhanced and decreased cell
IL-6, TNF-α, IL-12p70, CCL3, CCL4, and CXCL10) re-
for potential translation to liver cancer and other
surface expression, respectively. By extension, stimu-
sulted following the addition of each TLR7/8 agonist.
diseases.
lation of TLR7 and TLR8 elicited a modest increase
However, additive effects between CA9:GM-CSF and
+
in the costimulatory capacity of CD14 large APC in
the TLR agonists in enhancing growth factor secre-
allogeneic mixed lymphocyte response assays. Sig-
tion were minimal as each TLR agonist alone resulted
nificantly elevated levels of IL-1α, IL-1β, and MCP1-3
in maximal accumulation. Thus, in the ACI product,
in culture supernatant were consistent with APC acti-
agonists of TLR7/8 enhance T-cell activation and po-
vation. Collectively, antigen derived GM-CSF activity
larize T-cell responses to varying degrees dependent
is robust at activating APC and agonists of TLR7/8
upon receptor specificity.
can bolster this effect. But critically, using an HLA
Overall, agonists of TLR7/8 are compatible with the
class II restricted CA9-specific T-cell hybridoma re-
ACI platform for potentially enhancing antigen spe-
porter assay, it was imperative to stagger the addition
cific immunotherapy of cancer. In current experi-
of CA9:GM-CSF prior to TLR7 and TLR8 agonists in
ments, we are investigating the capacity of TLR7/8
order to maintain antigen uptake and peptide pres-
agonists to enhance cross-priming and generate cy-
entation. This suggests that GM-CSF activity initiates
totoxic T-cell responses.
2
1
Sipuleucel-T (Provenge®), indicated for the treat-
+
128
2
Simona Bot , Feng He , W. Gerald Newmin , Anand Ghanekar
cytokine induction without cytotoxicity. This is
129
079 | Improving Immunity
080 | Improving Immunity
mTOR inhibitor Rapamycin Enhances the Cancer Therapeutic
Potency of Naked RNA Vaccine
CpG Oligodeoxynucleotides enhances both humoral and cellular immune responses against FMDV in mice
1
1
1
1
2
1
2
2
3
3
Diken Mustafa , Kreiter Sebastian , Vascotto Fulvia , Selmi Abderraouf , Attig Sebastian ,
1
3
1, 2
Diekmann Jan , Türeci Özlem , Sahin Ugur Fuat Cem Yagci , Can Cokcaliskan , Musa Alkan , Bilgi Gungor , Mayda Gursel , Ihsan
1
Gursel 1
TRON - Translational Oncology at the University Medical Center of Johannes Gutenberg
­University, Mainz, Germany
1
Biotherapeutic ODN Research Lab., Department of Molecular Biology and Genetics, Bilkent
University, Ankara, Turkey
2
III. Medical Department, University Medical Center of Johannes Gutenberg
­University, Mainz, Germany
2
Department of Vaccine Research, Institute of FMD, Ankara, Turkey
3
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
3
Ganymed Pharmaceuticals, Mainz, Germany
Mammalian target of Rapamycin (mTOR) is a key
group. Moreover, Rapamycin treatment increased
Foot and Mouth Disease is one of the major con-
Our results indicated that formulations with CpG
metabolic kinase that regulates a variety of path-
the median survival rate (91 days compared to vac-
tagious and devastating viral diseases which is
ODN were induced significantly higher levels of
ways inside the cell. Recent reports have suggested
cination alone (46 days)) and delayed tumor ap-
caused by FMD virus (FMDV) of the cloven hoofed
total IgG and IgG2a antibodies either over oil-emul-
an additional role for mTOR as an intrinsic regula-
pearence (61.5 days compared to vaccination alone
animals. FMD outbreaks all around the world
sion formulated monovalent Serotype–O Ag or its
tor of memory T-cell formation. Treatment of mice
(34 days)). These findings underline the importance
caused serious economical losses during the last
free counterpart. Moreover, CpG ODN induced a
with the mTOR inhibitor Rapamycin was shown
of immunoregulatory strategies for the design of
century. Conventionally, inactivated FMDV is the
robust Th1-biased immune response as evidenced
to increase the number of memory precursors and
improved cancer vaccines.
major vaccine component used to control or eradi-
by increased IgG2a/IgG1>1 ratio. This increased
accelerated the transition from effector to memory
cate the disease. These vaccines have proven to be
Ab milieu was persistent over a course of 5 months.
cells. These studies used viral or bacterial infec-
an important component of control and eradication
The virus neutralization assays revealed that CpG
tion models and have not addressed the effect of
of the disease so far. However due to difficulties to
ODN added treatment groups developed much
mTOR inhibiton on the memory T-cell formation
grow certain serotypes and subtypes in cell culture
higher neutralizing antibody titers compared to
in the anti-tumor vaccination setting which is an
to get sufficient amount of antigen (Ag) for vaccine
non-CpG formulations. Consistent with anti-FMD-
important factor predicting the success of a cancer
production and the lack of longevity and rapidity
Ab findings, virus neutralization titer levels and
vaccine. In this study, we investigated the effect of
features of currently used vaccines, more rapid and
duration of neutralization were superior in CpG
Rapamycin on the potency of a naked RNA vaccine.
potent vaccination strategies are urgently needed.
groups.
C57BL/6 mice were immunized intranodally with
Synthetic oligodeoxynucleotides (ODNs) contain-
IFNγ is one of the major Th1 type cytokine which
naked RNA encoding for the immunodominant
ing unmethylated CpG bases due to their high im-
plays critical roles in the improvement of CD8
epitope of chicken Ovalbumin (SIINFEKL) and
munoadjuvant activity that promotes humoral and
T-cell responses. It has been reported that IFNγ
subsequently treated with Rapamycin (600µg/kg
cell mediated immunity is receiving great atten-
induced in vaccinated cattle is correlated with the
body weight) in the T-cell contraction phase for
tion. Several animal challenge studies and clini-
animal‘s ability to control the replication of FMD
three weeks. While the monitoring of SIINFEKL
cal trials indicated that co-administration of CpG
virus. Since mice injected with CpG ODNs induced
specific CD8 T-cells showed similar frequencies
ODNs with increased vaccine induced protection.
such high IgG2a/IgG1 ratios, we investigated
in the Rapamycin and control group, a higher fre-
The aim of this study is to develop CpG ODN adju-
whether CpG ODN including formulations can also
+
+
-
KLRG1 )
vanted FMD vaccine that confers long lasting im-
induce cell-mediated immunity. As an indicator of
were observed in mice received Rapamycin. This
munity and better protection against the disease.
cellular immune responses serum IFNγ levels of
group also exhibited more profound expansion in
6-8 week old female BALB/c mice (8-10/group)
mice have been measured by ELISA. Our results
response to rechallenge with SIINFEKL peptide.
were injected twice intraperitoneally at day=0 and
revealed that 24h after injection CpG containing
To address the question whether these effects of
15 either with licenced monovalent vaccine or free
formulations induced 1,5 to 2 fold more IFNγ in
Rapamycin can be translated into improved anti-
FMDV serotype-O antigen were combined with
serum which indicates the contribution of cell-me-
tumoral therapy, a therapeutic tumor experiment
CpG ODN (5ug/animal). Mouse sera were collected
diated immune response
using the B16-OVA melanoma model was per-
with 2 weeks intervals for 5 months. Serum O-spe-
In summary, this study demonstrated that CpG
formed. Analysis of tumor infiltrating cells re-
cific total IgG, IgG1, IgG2a antibodies (Ab) and IFNγ
ODN provided an Ag sparing effect while it
vealed a higher percentage of SIINFEKL specific
levels were determined by ELISA. Neutralization
achieved an enhancement both for humoral and
levels of immune mouse sera were determined by
cellular immune responses required to control
FMD virus neutralization assay.
better FMDV infection.
quency of memory precursors (CD127
+
CD8
T-cells and a lower frequency of myeloid
derived suppressor cells (MDSC) in the Rapamycin
130
+
131
081 | Improving Immunity
082 | Improving Immunity
Potent Stimulation of the Innate Immune System by Nucleic
Acid Based TLR Ligands Encapsulated in Nanoliposomes
CpG DNA containing nanoparticles as potential antitumor
agents
Banu Bayyurt, Ihsan Gursel
Gizem Tincer , Kutay Karatepe , Cevayir Coban , Ken Ishii , Mayda Gursel , Ihsan Gursel Biotherapeutics ODN Research Lab., Department of Molecular Biology and Genetics,
1
Biotherapeutic ODN Lab, Department of Molecular Biology and Genetics, Bilkent University,
Ankara, Turkey.
2
Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University,
Osaka, Japan.
3
Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Osaka, Japan.
4
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
1
Bilkent University, Ankara, TURKEY
132
1
2
2, 3
4
1
Nucleic acids with bacterial and viral origins are
Additionally, thioglycollated peritoneal exudate
Toll-like receptor 9 (TLR9) agonist CpG oligode-
Untreated or free ODN treated nude mice at the end of
recognized by toll like receptors (TLRs) that trig-
cells (PECs) were isolated and treated with differ-
oxynucleotides (CpG ODN hereafter) were used for
d=30 developed a tumor volume ~3757±1050 mm
gers mammalian innate immune system to secrete
ent liposome formulations in order to investigate
cancer immunotherapy of established hepatocellu-
and 1300±235mm respectively. A >95% of tumor
proinflammatory or inflammatory cytokines. Even
inflammasome inducing ability of unencapsulated
lar and tyhmoma tumor models.
size reduction was observed in mice treated with
though these ligands have a high potency to be
free liposomes or together with their cargo. The
Among many, induction of cytotoxic T-cells (CTL),
NP-ODN. Of note, a 40% complete regression was
used in clinical applications as a vaccine adjuvant
cells were studied by FACS, while the supernatants
improving antigen presenting cell (APC) functions
observed in NP-ODN treated group whereas there
or as an immunotherapeutic agent, the rapid clear-
were analyzed for IL1b production by ELISA.
and subversion of immunosuppressive microen-
was no complete tumor remission in any other
ance from the body by serum proteins, in vivo deg-
Our data revealed that when DCs were treated with
vironment generated by tumors are some of these
treatment groups.
radation via nucleases, consequently lead to poor in
liposomal formulations, maturation signals along
mechanisms mediated by CpG ODNs. Yet when
The antitumor effect of natural PS/CpG-ODN nano-
vivo stability and performance thus hampers their
weith co-stimulatory molecule expression of DCs
these agents were given in vivo, they are rapidly
complexes were more effective than untreated or
clinical applications.
were improved up to 50 fold compared to free CpG
cleared by nucleases, and could be adsorbed by
only PS treated C57BL/6 mice that was inoculated
Drug delivery systems are promising tools to in-
or pIC induced activations. The IL6, IL12 and IFNγ
circulating serum proteins hampering their in vivo
with EG7 cells. Almost >50% reduction in tumor
crease the stability of therapeutic agents and provide
production from splenocytes and DCs were signifi-
therapeutic applications.
size were observed in mice that had nanocomplex.
the targeting and internalization of the incorporated
cantly boosted by liposomes.
Modification of CpG ODNs into a more stable form
Starting from day 16 and onwards this profile re-
cargo. Among many, liposomes that are nano-sized
This improvement was highest for CpG-A loaded
critically improve its application in immunothera-
mained same until the cessation of experiment on
lipid vesicles are one of the most commonly used
anionic liposomes, whereas stealth
py against cancer.
d=30.
drug delivery vehicles. Due to safety and high en-
cationic liposomes were the best formulation for
We have designed a novel class of ODN, devoid of
Treating mice either with self-nanoparticle forming
trapment efficiency of bioactive agents liposomes
CpG-B type. Neutral and stealth cationic liposomes
polyGs that can undergo nanoparticle formation-
CpG ODN or PS/CpGODN nanocomplexes demon-
are one of the best candidates to be utilized in the
were very effective formulations for pIC mediated
designated as NP-ODN. In addition to that, CpG
strated a significant tumor regression in two differ-
encapsulation of very labile and valuable bioactive
cytokine production and DC maturation. Surpris-
ODN-natural polysaccharide nanocomplex formu-
ent established tumor models.
agents. In this study, different liposomes were used
ingly, formulations containing cholesterol were
lations were also characterized to be tested as ef-
The mechanism responsible from this effect
to encapsulate TLR ligands in order to understand
generally very effective inflammasome activators
fective anti-cancer agents to control established
could be attributed to a more pronounced natural
their immune stimulatory effects.
as evidenced by IL-1b production of stimulated
tumor growth.
killer cell activation in hepatocellular carcinoma
Mouse splenocytes and bone marrow derived mDCs
PECs.
HUH7, hepatocellular carcinoma and EG7 thymoma
model, and more effective APC and CTL activity in
were stimulated with different liposome formula-
In conclusion, it was demonstrated that liposome
xenografts were established in athymic nude and
thymoma model. Involvement of various cell types
tions with varying physiochemical features loaded
formulations are efficient vehicles to increase the
C57BL/6 mice (5-10/group), respectively. Three
regulating CpG-nanoparticle mediated tumor re-
with CpG (A or B-types) and pI:C (TLR9 and TLR3
immune stimulation potency of TLR ligands by
doses of 100µg NP-ODN or PS/CpG ODN nanocom-
gression is under investigation.
ligands, respectively). Production of proinflamma-
increasing DC maturation, pro-inflammatory cy-
plexes on alternating days were intratumorally (or
tory cytokines were analyzed by ELISA and the
tokine secretion and inflammasome activation.
peritumorally) administered following palpable
maturation level and expression of co-stimulatory
tumor formation of mice and tumor dimensions
molecules of DCs was studied by FACS.
were recorded daily and calculated as in mm .
3
3
3
Acknowledgements: IG received grant supports from
TUBITAK, SBAG (Grant #: 108S316, 111S216) and SANTEZ2009-2-STZ-00448.
133
083 | Improving Immunity
084 | Improving Immunity
Potentiating the immunostimulatory properties of CpG oligodeoxynucleotides: aiming to develop a better vaccine adjuvant
A novel ODN delivery platform: CpG-ODN-loaded exosome
nanovesicles
1
2
2
1
Bilgi Gungor , Fuat Cem Yagci , Ihsan Gursel , Mayda Gursel Tamer Kahraman, Gozde Gucluler, Ihsan Gursel
1
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
2
Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
Unmethylated CpG DNA is recognized by TLR9
CpG or CpG/LL-37, suggesting a more potent Th1
Exosomes are naturally occurring, membranous
of cellular internalization. Data revealed that K3
expressed by B lymphocytes, dendritic cells (DC)
skewing potential.
nanovesicles of 40-100 nm in diameter. They arise
exosomes loaded cells were 80% positive for both
and macrophages. Synthetic (ODN) containing
Next, 6-8 week old BALB/c mice (5/group) were im-
from the endocytic cellular pathway through three
Cy5 and SP-DiOC signal. RAW cells were found to
unmethylated CpG motifs duplicate the ability of
munized twice (days 0 and 15) with 5X lower dose
stages: (i) formation of endocytic vesicles ii) mul-
be 100% positive for Cy5 and SP-DiOC in the case of
bacterial DNA to stimulate the innate immune
of the optimal licenced monovalent inactivated
tivesicular bodies (MVBs) formation in the cytosol
D35-loaded exosomes. Co-localization of exosomes
system via TLR9. Murine models indicate that the
FMD vaccine mixed with i) CpG ODN, ii) CpG ODN/
and (iii) fusion of the MVBs with the plasma mem-
and ODN signals in the cells were confirmed by
innate immune response elicited by CpG ODN can
anti-microbial cationic peptide LL-37 complexes or
brane to release their nanovesicular cargoes (1, 2).
confocal microscopy. Supernatants from CpG ODN
be harnessed to help eliminate cancers, prevent al-
iii) CpG ODN/cationic peptide Tat complexes. CpG
As well as functioning as natural vectors of inter-
loaded nanovesicle treated cells were used to detect
lergic reactions and boost the immunogenicity of
ODN dose/mice was adjusted so that each animal
cellular signaling within a given tissue or between
TNFα, and IL6 secretion profiles by ELISA.
vaccines. Numerous pre-clinical studies show that
received a 5X lower dose (2 mg) of ODN than the
differenT-cells and tissues, exosomes could be ex-
This data implicated that K and D-type CpGODNs
CpG ODN are effective as vaccine adjuvants and
optimal adjuvant dose in mice (10 mg). The pre-
ploited for the delivery of therapeutic cargoes (3).
can be effectively encapsulated into exosomes by
that they synergistically enhance tumor regression
cursor frequencies of serotype O specific IgG1 or
To explore the therapeutic potential, we investi-
lyophilization. Encapsulation of these particles
when used in combination with surgery, chemo-
IgG2a secreting cells were determined using a lim-
gated whether cell line-derived exosomes could be
into exosomes enhanced their stimulatory activity
therapy and/or radiotherapy. Based on such find-
iting dilution assay. Antigen specific proliferation
loaded with ss/ds DNA or RNA. The nano-delivery
as evidenced by their increased IL-6 and TNF-α se-
ings, more than a dozen clinical trials utilizing CpG
of lymphocytes was assessed in inactivated virus
vehicle is expected to provide better protection of
cretion by RAW264.7 cells. This effect was dose de-
ODN have been conducted. Unfortunately, clinical
stimulated cultures using CFSE dye dilution assay.
the cargo, more effective delivery to immune sites
pendent for both K3 and D35 containing exosomes.
results suggest that CpG ODN are somewhat less
IgG1 secreting precursor frequencies of animals im-
while facilitating better internalization that lead to
Internalization kinetics and magnitude of ODNs
potent in humans than in rodents. Herein we sum-
munized with the FMD vaccine adjuvanted with
enhanced activity.
when loaded into exosomes were much higher than
marize our efforts to potentiate the immunostimu-
CpG, CpG/LL-37 and CpG/Tat were determined to
Exosomes were isolated from RAW264.7 superna-
free ODN.
latory activity of CpG ODN by simple complexation
be 1/30,000, 1/110,000 and 1/100,000, respective-
tants by i) differential centrifugation, ii) filtration
Collectively, our findings strongly demonstrated
with the cell penetrating peptide Tat to generate
ly, whereas the frequencies of IgG2a secreting cells
and iii) ultra-centrifugation. Purified exosomes
that exosome-loaded DNA led to formation of ef-
a potent adjuvant formulation that specifically in-
were 1/88,000, 1/52,000 and 1/12,000, indicating
were loaded with Cy5- labeled K3 and D35 CpG
fective nanovesicle drug delivery system.
crease the precursor frequency of IgG2a secreting
that CpG/Tat preferentially expands the popula-
oligos via dehydration-rehydration method and
This technique promises to offer a biocompatible
cells in mice immunized with the inactivated foot
tion of IgG2a secreting cells. Since evidence sug-
labeled with lipophilic dye, SP-DiOC(18). SP-DiOC
and personalized therapeutic approach with a great
and mouth disease (FMD) vaccine.
gests that tumor regression in mice is associated
labeled, CpG ODN loaded exosomes were incubated
potential to overcome the poor performance mainly
CpG ODN/Tat peptide complexes were prepared at
with a sustained and predominant IgG2a antibody
with RAW 264.7 cells at 10:1 ratio (Exosome:Cell)
due to pre-mature elimination of synthetic particles
different molar ratios (1:2, 1:4, 1:8, 1:16) and the
response, and tumor progression with a mixed
for 24h for cytokine production assays or incubated
since they are recognized as non-self upon in vivo
formulation that induced a robust type I interfer-
IgG1/IgG2a response. Our results suggest that the
for several hours for confocal studies. For exosome
administration.
on production in human PBMC was selected for
immunostimulatory properties of CpG/Tat could
internalization studies, cells were analyzed by
studies in mice. Stimulation of mice splenocytes
be more suitable than CpG ODN in development of
FACS or by confocal microscopy. Exosome treated
with CpG ODN, CpG/Tat or CpG/LL-37 (a cationic
specifically anti-cancer vaccines.
cell supernatants were used for cytokine ELISA
assays.
antimicrobial peptide withouT-cell penetration
property) at a dose of 0.3, 1 or 3 mM showed that
CpG/Tat induced a 2X increase in IFNγ production
at all doses tested against the maximum dose of
134
Acknowledgment: This work was supported by a TUBITAK
grant (111S151).
Different CpG DNA loaded SP-DiOC labeled exosomes were (i.e. Exo(K3-Cy5)SP-DiOC or Exo(D35-
References: [1] Trajkovic K, Hsu C, Chiantia S, Rajendran
L, et al. 2008. Science 319:1244–7. , [2] Chaput N, Théry C.
2011. Semin Immunopathol. 33:419-40. , [3] Lakhal S, Wood
MJ. 2011. Bioessays 33:737-41.
Acknowledgment: This work was supported by a TUBITAK
SBAG grant #: 111S316.
Cy5)SP-DiOC) analyzed by FACS to assess the level
135
085 | Improving Immunity
086 | Improving Immunity
Bacteriocin DNA nanocomplexes as immunotherapeutic carriers
Alternative approaches to improve immunity against cancer
and infectious diseases: development of nucleic acid loaded
nanocarrier systems
1
1
2
1
Fuat Cem Yagci , Gozde Gucluler , Fadime Kiran , and Ihsan Gursel 1
Biotherapeutic ODN Research Lab, Department of Molecular Biology and Genetics, Bilkent University,
2
Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey
Synthetic oligodeoxynucleotides (CpG-ODNs) and bac-
to 1:2, 1:4, 1:8, ODN:Bacteriocin ratio (w/w). Single-
The immunogenicity of a delivery formulation is closely
PS/TLR9L nanocomplexes were tested in established
dependent on effective internalization of immunogen
thymoma model. Data revealed that PS/CpGODN nano-
and adjuvant by the innate immune cells. These effects
complex led to a more effective anti-tumor activity than
untreated or only PS treated C57BL/6 mice that was
terial DNA containing unmethylated CpG dinucleotides
cell splenocyte suspensions were prepared (5x10 /mL)
are optimized by maintaining close physical contact
(CpG motifs) are clinical candidates as anti-cancer
and added to each well.
between the adjuvant and the antigen. Moreover, sus-
inoculated with EG7 cells. Starting from post treatment
agents, immune adjuvants, anti-allergens, and stand
Three different CpG-ODN:bacteriocins complexes (1:2,
tained and simultaneous release of antigen and ad-
at day 16, more than 50% reduction in tumor sizes were
alone immunoprotective agents. Two different classes
1:4, 1:8) in four different concentrations (3, 1, 0,3 and
juvant to relevant antigen presenting cells mediated
obtained in mice that had nanocomplex. This profile re-
of CpG-ODNs were identified for clinical applications.
0,1µM) were used to stimulate the cells for 24 hr. Super-
by a depot carrier system is one of the hallmarks of
mained same until the cessation of experiment at d=30.
B-Class ODNs, also known as K-type CpG ODN, have
natants were collected and stored for cytokine ELISA
generating long lasting effective immunity. Endosome-
II. Vaccine development by liposomes: Liposome encap-
phosphorothioate backbones and potent B- cell acti-
at -20°C.
associated TLRs recognizes microbial nucleic acids (ss/
sulation is a powerful tool to increase in vivo stability
vators. A-Class ODNs (or D- type ODNs) have mixed
Secretion of IL-6 and IL-12 into culture supernatants
ds DNA or RNA). Clinical applications of these ligands
as well as enhancing internalization of its cargo to rel-
backbone and flanking G-runs at their 3`and 5`-ends.
were determined by ELISA. Concentrations were calcu-
were significantly hampered due to their pre-mature
evant immune cells. We established that encapsulat-
This class mainly triggers pDC to secrete IFNα, an im-
lated from standard curves generated by use of known
in vivo digestion by nucleases or rapid clearance due to
ing CpG motifs in different nanoliposomes (~100nm)
portant anti-viral cytokine. However, clinical trials re-
amount of recombinant mouse cytokines. All ELISA
circulating serum protein adsorption.
having different physicochemical properties altered
vealed that native CpG-ODNs` activities are not at the
assays were performed in triplicate for each groups.
This presentation will address our recent efforts on how
not only encapsulation efficiency, but also the release
therapeutic level, leading to failure of the ongoing trials.
Although bacteriocins were isolated from different
to formulate CpG ODNs and other related nucleic acid
and delivery rates that ultimately impacted in vitro and
Therefore, several different approaches have been pro-
bacterial cultures our data suggested that they are not
based TLR ligands able to yield more effective immuno-
ex-vivo cytokine production rates and types. Moreover,
posed in order to alleviate these undesirable side-effects
inducing any immune response by themselves. Upon
therapeutic nanocarrier systems. In brief, I will mainly
different liposomes encapsulating CpG ODN signifi-
(i.e. nanoliposomes, or amphipfilic biodegradable mac-
complexation with CpG-ODNs, the nanocomplexes
focus on the development of nucleic acid containing
cantly increased Th1-biased cytokine and chemokine
romolecules [1,2]). Simpler complexation compounds
induced significantly much higher IL-6 and IL-12 pro-
nanoliposomes, polysaccharide nanocomplexes, modi-
gene transcripts. Additional studies demonstrated that
that provide increased stability, retained activation and
duction from mouse splenocytes. Enterocin A was
fied montanide nano-emulsions, self-aggregating den-
co-stimulatory and surface marker molecules on B-, DC,
improved internalization of labile molecules such as
found to be more effective on IL-6 secretion when used
drimeric DNA-nanoparticles, and cell derived exosome
and Macrophages significantly upregulated upon lipo-
CpG motif expressing ODNs, if available in bulk and
together with D or K-ODNs as compared to pediocin
nanovesicles as effective nucleic acid carriers against
some/CpG injection. Finally, co-encapsulating model
can be obtained very cheaply are very suitable carri-
AcH/PA-1. Interestingly, when IL-12 induction profiles
viral or bacterial infections as well as candidate agents
antigen ovalbumin with CpG ODN adjuvant in nanoli-
ers. Cationic peptides (enterocin A and pediocin AcH/
were investigated, data implicated that both bacteri-
for anti-cancer immunotherapy. Some exemplary ap-
posomes profoundly augmented Th1 and cell mediated
PA-1) isolated from different lactic acid bacteria (LAB)
ocins were able to pronounce the effect of D but not
proaches to improve in vivo performance of TLR-based
anti-ova specific immune response. This work estab-
are such compounds, since they can interact with CpG-
K–type CpG ODNs (data not shown).
immuntherapeutics are covered below.
lished an unappreciated immunoregulatory property
ODNs to generate stable nanocomplexes. The bacteri-
When taken together present data demonstrated that
I. Polysaccharide based nanocomplexes: Following ex-
of nanoliposomes mediating immunity against protein
ocins produced by LAB, are ribosomally synthesized
bacteriocin/DNA nanocompexes significantly aug-
ploration of immunostimulatory properties of mush-
antigen and could be harnessed to design more effec-
antimicrobial peptides. The peptide is heat-stable, and
mented immunostimulatory potential of different
room derived polysaccharides (PS), stable i) PS/pIC, ii)
tive therapeutic vaccines or stand alone immunopro-
cationic [3]. Here we showed that; two candidate bac-
classes of CpG motifs. These results strongly sug-
PS/CpGODN, and iii) PS/R848 nanocomplexes around
tective agents targeting infectious diseases, as well as
teriocins, namely, i) enterocin A and ii) pediocin AcH/
gested that bacteriocins are suitable carriers for labile
100-150 nm in size were prepared. PSs were selectively
cancer or allergy.
PA-1 complexed with different CpG ODNs led to supe-
nucleic acid based agonists nanocomplaxation, and
engaged by cells expressing TLR2 and initiated NFκB
Collectively, development of techniques that provide
rior activity over free ODN counterparts.
could be harnessed to improve their in vivo efficacy
dependent signalling cascade leading to a Th1-biased
stable encapsulation and or complexation of nucleic
for immunotherapeutic applications.
cytokine/chemokine secretion in addition to bactericid-
acid based bioactive drugs in a suitable carrier/depot
control ODNs in which the CpG motif was methyl-
al nitric oxide (NO) production by macrophages. More-
system is an effective approach to improve in vivo per-
ated or inverted, were used as CpG-A and CpG-B type
References:
over, cells treated with nanoparticles led to synergistic
formance and serves to broaden the immunotherapeu-
ODNs, respectively. In this study, Enterocin A, pro-
(1) I. Gursel, J.Immunol, 2001,
IL6, production and upregulation of TNFα, MIP3α, IFNγ
tic spectrum of TLR based nucleic acid ligands.
duced by infant isolate of Enterococcus faecalis OZV
(2) G. Tincer, Biomaterials, 2011,
and IP10 transcript expression. In mice, PS-Ag-Nucleic
and Pediocin AcH/PA-1, produced by breast milk
(3) R.W.Jack, Microbiol Rev., 1995.
Acid formulation surpassed antigen-specific IgG re-
isolate of Pediococcus pentosaceus OZF were used as
Acknowledgements: Grants from TUBITAK/SBAG (Project#:
111S216, and 108S316)is appreciated.
Acknowledgment: Part of this work was supported by
TUBITAK grants (108S316, and 111S236) and SANTEZ grant
(STZ-2009-2-00448) to IG. Contributions from the old and
current members of IG Lab is greatly appreciated.
Immunostimulatory ODN-D35 and ODN-K3, and their
bacteriocin sources. The complexations were adjusted
136
Ihsan Gursel
Biotherapeutic ODN Res Lab., Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
6
sponses when compared to either PS-Ag or nucleic acid
ligand-Ag mediated immunity. The antitumor effect of
137
087 | Immunomonitoring
Divpenia (low TCR diversity) and lymphopenia as prognostic
factors of OS in metastatic breast cancer
Immunomonitoring
138
1
4
4
3
1
1
M. Manuel , O. Trédan , T. Bachelot , G. Clapisson , A. Courtier , G. Parmentier ,
1
1
1
1
5
5
4
T. Rabeony , A. Grives , S. Perez , JF. Mouret , D. Perol , S. Chabaud , I. Ray-Coquard ,
4
4
6
2, 3
2
1
I. Labidi-Galy , P. Heudel , JY. Pierga , C. Caux , JY. Blay , N. Pasqual , C. Ménétrier2, 3
Caux
1
ImmunID Technologies, CEA, 17 rue des Martyrs, 38054 Grenoble, France
2
Team 11 "Therapeutic targeting of tumor cells and their immune environment" CRCL INSERM
U-1052/CNRS 5286, 28 rue Laennec 69008 Lyon, France
3
Innovation in Immunotherapy and Immuno-monitoring (PI3), Centre Léon Bérard, 28 rue
Laennec 69008 Lyon, France
4
Department of medical oncology, Centre Léon Bérard, 28 rue Laennec, 69008 Lyon, France
5
Biostatistic Unit, Centre Léon Bérard, 28 rue Laennec 69008, Lyon, France
Rationale: Recent works have highlighted that
penia (<1 Giga/L) was associated with shorter OS
monitoring lymphocyte count in peripheral blood
for cohort B. The combination of low TCR diver-
before any chemotherapy had a prognostic value on
sity with lymphopenia (called lympho-divpenia)
patient survival in metastatic breast cancer. Lym-
was associated with poor OS compared to patients
phopenia (<1 Giga/L) prior to treatment is a predic-
with either diversity >33% or lymphocyte count
tive factor for toxicity and death in metastatic solid
≥1 Giga/L or both, in cohort A (median OS: 7.6 vs
tumors. On the other hand, TCR diversity is direct-
24.5 months, log rank p.value =0.0006) and cohort
ly related to antigen recognition and is required
B (median OS 10.6 vs 22.9 months, log rank p.value
for mounting an efficient immune response. Com-
=0.0035). In a multivariate analysis, including
binatorial diversity of T-cell Receptor -beta chain
other significant clinical factors from the univari-
(TCR-β), as a measure of T-cell repertoire diversity,
ate analysis (Performance Status, liver metasta-
was investigated and tested either alone or in com-
sis, hemoglobin) lympho-divpenia was found to
bination with lymphopenia as a prognostic factor
be an independent prognostic factor in the pooled
at diagnostic for overall survival (OS) in metastatic
cohort (A+B) (p=0.005) along with triple negative
breast cancer (MBC) patients.
tumors (p=0.011) and hemoglobin level (11.5 g/dL)
Patients and methods: Using semi quantitative
(p=0.009).
multiplex PCR, the V-D-J combinatorial diversity
Conclusion: The NDL score, combining lymphope-
of the TCR was measured on cryo-preserved blood
nia and reduced TCR diversity, is a strong prognos-
samples from 2 cohorts of MBC patients before the
tic factor and could help improving care quality
initiation of the fi rst line chemotherapy: a devel-
of MBC patients. Our results highlight the exist-
opment cohort (cohort A, n=66) and a validation
ence of an immunodeficient sub-population of MBC
cohort (cohort B, n=67). A prognostic score, called
patients associated with a shorter life expectancy.
NDL (Number & Diversity of Lymphocytes) com-
A clinical study is ongoing in order to determine
bining lymphocyte count and TCR diversity was
whether that subset of patients could benefit from
evaluated. Univariate and multivariate analysis of
a therapeutic adaptation combining chemotherapy
prognostic factors for OS were performed in both
and IL-7, a cytokine that promotes immuno-recon-
cohorts.
stitution through peripheral lymphocytes T prolif-
Results: TCR diversity ≤33% (divpenia) was as-
eration and thymic activation.
sociated with a reduced OS in cohort A. Lympho139
088 | Immunomonitoring
089 | Immunomonitoring
T-cell recognition of Tumor-associated antigens in patients with
preinvasive lesions of the breast
Antibodies against CTL epitopes from tumor-associated antigens were widely detectable in humans: potential prognostic
significance in cancer patients
1
1
1
1
1
1, 2
1
3
3
1
Anna-Lena Krause , Yingzi Ge , Andreas Bonertz , Joanna Förster , Christoph Domschke ,
2
1
Florian Schütz and Philipp Beckhove
Satoko Matsueda , Nobukazu Komatsu , Kenichi Kusumoto , Shintaro Koga , Shigeru Yutani ,
1
4
1
1
Shigeki Shichijo , Takashi Mine , Kyogo Itoh , Tetsuro Sasada
1
1
Det. of Immunology and Immunotherapy, Kurume University School of Medicine, Kurume,
Japan
2
Cancer Vaccine Division, Research Center of Innovative Cancer Therapy, Kurume University,
Kurume, Japan
3
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume,
Japan
4
Multidisciplinary Cancer Center, Kurume University School of medicine, Kurume, Japan
2
Division of Translational Immunology, Tumor Immunology Program, German Cancer Research
Center (DKFZ), National Center for Tumor Diseases (NCT), INF 460, 69120 Heidelberg, Germany
Department of Gynecology and Obstetrics, University Hospital of Heidelberg, Voßstraße 9,
69115 Heidelberg, Germany
140
Breast cancer cells are known to frequently (over)
linked immunospot assay (ELISpot). Therefore, we
Purpose: To investigate humoral responses to CTL
express several well-characterized tumor-associ-
stimulated peripheral blood T-cells with autologous
epitope peptides derived from tumor associated an-
ated antigens (TAAs) such as carcinoembryonic
dendritic cells pulsed with long synthetic peptides
tigens (TAA) in healthy donors (HD) or patients
antigen (CEA), MUC-1, Her-2/neu, members of the
bearing a 50 amino acid long sequence of CEA,
with various types of diseases.
MAGE family, Mammaglobin and Heparanase.
MUC-1, Her-2/neu, MAGE A3, Mammaglobin1 or
Experimental design: Plasma or sera were collect-
Spontaneous T-cell responses against tumor-as-
Heparanase1. These peptides contain predicted
ed from HD, patients with hepatitis C virus (HCV)
sociated antigens (TAAs) are frequent in patients
ligands for the most common HLA molecules of
infection, influenza virus infection, rheumatoid
with invasive breast cancer, but rare in healthy in-
the german polulation. Each experiment was per-
arthritis, IgA nephropathy, hematopoietic malig-
dividuals. Despite the presence of cytotoxic effec-
formed before and after depletion of Tregs.
nancy, and non-HCV hepatocellular carcinoma.
tor cells, the antitumor-response can be inhibited
We show that in peripheral blood of DCIS patients,
Bead-based multiplex assay (Luminex system) was
by regulatory T-cells (Tregs), which accumulate
the frequency of TAA-specific T-cells is significant-
used to measure immunoglobulins (Igs) to each of
in the tumor. However, up to now, it is unknown
ly higher than in healthy donors and does not differ
31 different peptides from TAA capable of inducing
in which stage of the cancerogenesis TAA-specific
from patients with invasive breast cancer. While
CTL responses.
T-cell immunity emerges and if the induction of
Treg depletion significantly increases the in vitro
Results: IgGs specific to the most of 31 peptides
tumor-associated Treg is an early or late process.
responsiveness in invasive breast cancer patients,
tested were detectable in the majority (>50%) of
Since the introduction of the Mammography
we could not observe this effect in DCIS patients.
HD. Age-depended decrease was observed on the
screening program in Germany, increasing prein-
Our data suggest that already in a preinvasive state,
total sums of anti-peptide IgMs, whereas those of
vasive lesions of the breast are detected. The most
when malignanT-cells are bounded to the ducts,
anti-peptide IgGs increased in elder HD. Patients
common preinvasive lesion, the ductal carcinoma
breast carcinomas appear to be immunogenic. Fur-
with HCV or influenza infection had higher sums
in situ (DCIS), is characterized by proliferation of
thermore, the induction of Tregs, capable of inhib-
of IgGs as compared to HD. Patients with hemat-
malignant epithelial cells within the breast paren-
iting the antitumor-response, might occur later in
opoietic malignancy showed higher sums of anti-
chymal structures, with no evidence of invasion
the carcinogenesis. Currently, we are investigating
peptide IgMs and lower sums of anti-peptide IgGs,
across the basement membrane. If left untreated,
the immunogenicity of benign proliferative lesions
whereas the sums of both of anti-peptide IgMs and
up to 50% transform into an invasive carcinoma
of the breast.
anti-peptide IgGs were higher in HCC patients. The
within 10 years.
total sums of anti-peptide IgG1 were well correlated
Our aim was to investigate the T-cell-reactivity of
with overall survival in both malignant diseases.
DCIS patients against TAAs shown to be relevant in
Conclusions: Humoral responses to CTL epitope
invasive breast cancer and the impact of regulatory
peptides derived from TAA were detectable in
T-cells on this responsiveness.
humans. Our results could provide new insight for
To investigate the functional activation of the T-cell
better understanding and clinical significance of
pool by TAAs, we performed an IFN-γ Enzyme-
humoral responses to TAA-derived peptides.
141
090 | Immunomonitoring
091 | Immunomonitoring
Reliable assay for monitoring CMV-specific T-cell immunity
­following Allogeneic Hematopoietic Cell Transplantation
Expression of Foxp3 in Colorectal Cancer but not in Treg Cells
Correlates with Disease Progression in Patients with Colorectal
Cancer
1
1
2
2
1
1
2
2
1
Tina Jakobsen , Liselotte Brix , Dalin Pan , George Chen , Charlotte Halgreen ,
2
2
1
2
Theresa Hahn , Philip McCarthy , Henrik Pedersen and Paul K Wallace Martin Gasser , Tanja Grimmig , Maria Lazariotou , Christoph Thomas Germer ,
2
Ana Maria Waaga-Gasser 1
1
Department of Surgery I, University of Wuerzburg, Germany
2
Department of Surgery I, Molecular Oncology and Immunology, University of Würzburg,
­Germany
Immudex, Copenhagen, Denmark. 2Roswell Park Cancer Institute, Buffalo, New York, USA
Cytomegalovirus (CMV) infects and establishes
patients. Furthermore, in some recipients receiv-
Regulatory T-cells (Treg) expressing the transcrip-
persistent lifelong infections in 50-85% of adults.
ing transplants from HLA-mismatched donors we
tion factor Forkhead-Box-Protein P3 (Foxp3) have
Reactivation of the virus is a frequently occurring
could measure CMV-specific T-cells restricted by
been identified to counteract anti-tumor immune
complication of immunosuppression following
donors HLA and not recipients HLA, indicating that
responses during tumor progression. Besides,
transplantation and can significantly contribute to
adoptive transfer of CMV-specific T-cells can occur
Foxp3 presentation by cancer cells itself may also
morbidity and mortality in such patients.
with alloHCT.
allow them to evade from effector T-cell responses,
Reconstitution of CMV-specific T-cell immunity
This study demonstrates that CMV Dextramers are
resulting in a survival benefit of the tumor. For
after allogeneic hematopoietic cell transplantation
reliable reagents that can be used to monitor re-
colorectal cancer (CRC) the clinical relevance of
(alloHCT) has previously been quantified using
constitution of CMV immunity post-alloHCT, and
Foxp3 has not been evaluated in detail. Therefore
CMV tetramers, and shown to be a valuable aid
shows that adoptive transfer of anti-CMV immunity
the aim of this study was to study its impact in
in predicting patients at risk of developing CMV
can be quantified.
colorectal cancer (CRC).
reactivation. We have developed an assay for quan+
Gene and protein analysis of tumor tissues from
tifying CMV-specific CD8 T-cells using CMV-spe-
patients with CRC was performed to quantify the
cific Dextramers. Dextramers are MHC multimer
expression of Foxp3 in tumor infiltrating Treg and
reagents that are used in flow cytometry to detect
colon cancer cells. The results were correlated with
antigen-specific T-cells in the blood. Dextramers
clinicopathological parameters and patients overall
have much higher resolution than conventional
survival.
MHC multimers like Tetramers, and thus provide
Serial morphological analysis demonstrated Foxp3
a more reliable means for identification of antigen-
to be expressed in cancer cells. Moreover, high
specific T-cells.
Foxp3 expression of the cancer cells was associ-
We here show that the CMV Dextramer assay in-
ated with poor prognosis compared to patients with
cluding 7 alleles (HLA-A*01,-A*02, A*03, A*24, B*07,
low Foxp3 expression. In contrast, low and high
B*08 and B*35) has high specificity and sensitivity
Foxp3 level in tumor infiltrating Treg cells demon-
and accurately enumerates CMV-specific T-cells in
strated no significant differences in overall patient
both healthy donors and alloHCT patients, with a
survival. Our findings strongly suggest that Foxp3
lower detection limit of 0.08 cells/µl. The assay is
expression mediated by cancer cells rather than by
highly reproducible with low intra and inter assay
Treg cells contribute to disease progression in pa-
variation.
tients with CRC.
Using the CMV Dextramer assay we were able to
quantify reconstitution of CMV T-cell immunity
post transplantation at day 30, 100, and 365 in 89
142
143
092 | Immunomonitoring
093 | Immunomonitoring
Molecular signature of virus-specific T-cell polyfunctionality
Activation and frequency of Myeloid Subsets in peripheral
blood is associated with clinical outcome in prostate cancer patients treated with Prostate GVAX and ipilimumab
1
2
2
Yen-Ling Chiu , Jonathan Schneck , Mathias Oelke 1
Johns Hopkins University School of Medicine, Department of Pathology, Institute of Cell Engineering, Baltimore MD,USA
2
Immunology Graduate Program, Johns Hopkins University
1
2
2
1
1
3
S.J.A.M. Santegoets , Anita G.M. Stam , R.J. Scheper , S.M. Lougheed , H. Gall , K. Jooss ,
3
3
4
4
1
1
N. Sacks , K. Hege , I. Lowy , J.-M. Cuillerot , W.R. Gerritsen , A.J.M. van den Eertwegh ,
1
and T.D. de Gruijl .
1
2
Departments of Medical Oncology Pathology and of the VU Medical Centre, Amsterdam, Neth3
4
erlands; Cell Genesys Inc., San Francisco, CA; Medarex, Bloomsbury, NJ/Bristol-Myers Squibb
Company, Wallingford, CT
T-cell polyfunctionality has been shown to be
Blockade of the CTLA-4 immune checkpoint can
activation and recruitment to the vaccination sites.
crucial in protective immunity. However, the mo-
enhance anti-tumor responses and prolong sur-
Treatment-induced activation of BDCA1/CD1c
lecular mechanism controlling T-cell polyfunction-
vival, but it can also lead to the development of
cDC and 6-sulfo LacNAc
ality have not yet been determined. Specifically
severe and potentially life-threatening immune-
associated with significantly prolonged over-all
the genetic control linking antigen doses to poly-
related adverse events (IRAE). To avoid unneces-
survival (OS). In contrast, increased frequencies of
functionality has not been explored. In this study,
sary exposure to this risk, it is essential to identify
CD11b CD14-CD15
we found that human antigen specific CTL expan-
biomarkers that correlate with clinical activity and
suppressor cells (MDSC) and high pre-treatment
sion induced by high dose antigenic stimulation is
can be used to recognize and select patients that
levels of CD14 HLA-DR
associated with CTL with lower polyfunctionality
will benefit from immune checkpoint blockade. We
associated with reduced OS.
and higher inhibitory receptors expression, while
therefore performed extensive immune monitoring
Together with similar analyses of T-cell subsets,
optimal doses of antigen stimulation results in
in a phase I/II dose escalation/expansion trial of
these studies have yielded an immune profile with
higher CTL polyfunctionality and lower level of
combined Prostate GVAX (a GM-CSF-secreting al-
predictive value for clinical outcome. The profile
inhibitory receptors. Notably, we found that even
logeneic prostate cancer vaccine) and antiCTLA-4/
is characterized by pre- or on-treatment activation
in an artificial APC system with only signal 1 and
ipilimumab immunotherapy in patients with Cas-
of immune effector subsets and low frequencies of
signal 2, antigen dose still directly mediated CTL
tration Resistant Prostate Cancer (CRPC). Here we
regulatory/suppressive subsets. It may thus provide
polyfunctionality independent of inhibitory recep-
report on the effects of the treatment on circulat-
a potentially useful tool for patient selection and
tor signaling. The molecular signature associated
ing myeloid cells and the identification of potential
should be validated as such in other patient groups
with high CTL polyfunctionality is identified by
myeloid-related biomarkers.
treated by antiCTLA-4 blockade.
genomic microarray. These findings are thus im-
The GVAX/ipilimumab combination was clini-
portant framework for vaccine development.
cally active with PSA declines of more than 50%
+
+
+
+
+
inflammatory DC was
granulocytic myeloid-derived
lo/-
monocytic MDSC were
in 5, and PSA stabilizations in 12 of 28 patients.
Regressing bone and lymph node metastasis were
observed in 2/5 PR patients. Flowcytometric
monitoring of myeloid subsets in peripheral blood
before and after Prostate GVAX/ipilimumab treatment revealed some striking differences between
patients who benefited from therapy and patients
who did not. Significant treatment-induced decreases of conventional and plasmacytoid Dendritic Cell subsets (cDC and pDC, respectively) were
observed, which were paralleled by increased DC
144
145
094 | Immunomonitoring
095 | Immunomonitoring
CD8+ T-cells specific for peptides encoded by large T and small
T antigen from Merkel cell polyomavirus is detected in merkel
cell carcinoma patients and not healthy individuals
No correlation between spontaneous tumor antigen-specific
T-cell and antibody responses in the majority of primary melanoma patients
1
1
2
3
1
2
3
1
Rikke Lyngaa , Charlotte Albæk Thrue , Paul Nghiem , Jürgen. C. Becker , Per Thor1
1
Straten , Sine Reker Hadrup Christina Pfirschke , Christoffer Gebhardt , Inka Zörnig , Ludmila Umansky , Alexander
2
3
1
Enk , Dirk Jäger , Philipp Beckhove 1
Center for Cancer Immune Therapy, CCIT. Department of Hematology, University Hospital Herlev, Herlev, Denmark
1
Division of Translational Immunology, German Cancer Research Center (DKFZ), Heidelberg,
Germany
2
Departments of Medicine/Dermatology, Pathology, University of Washington, Fred Hutchinson Cancer Research Center, Seattle Cancer Center Care Alliance, Seattle, WA 98109, USA
2
Department of Dermatology, University of Heidelberg, Heidelberg, Germany
3
Department of Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany
3
General Dermatology, Medical University of Graz, Graz, Austria
Several types of cancers are known to have a viral
healthy donors by MHC multimer staining, combi-
Tumor-reactive memory T-cell (TC) responses can
Tyrosinase, MDM2, MAGE-A1, p53, MIF, RAB38,
origin. These include cervical cancer induced by
natorial color-encoded to allow parallel detection
be involved in durable prevention of tumor recur-
gp100, GAGE-1, TRP2, NY-ESO-1, MAGE-C2) and
Human Papilloma Virus and Kaposi Sarcoma induced
of T-cells specific for this large library of peptides.
rence and metastasis formation. Spontaneously
IgG control antigen. In addition, corresponding
by Karposis Scarcoma associated herpes Virus. A
Due to the low frequency of MCV specific CD8 T-
induced TC responses are present in patients with
spontaneous Ab responses were analyzed.
new member of the virus induced cancers was re-
cells in peripheral blood we performed a parallel en-
many tumor entities, including malignant mela-
In the majority of MM patients, independent of the
cently discovered, the Merkel Cell Carcinoma (MCC).
richment of all peptide reactive T-cells by magnetic
noma (MM), and provide a repertoire of functional
HLA-type, polyvalent preexisting TAA-reactive TC
Merkel cell carcinoma is a rare and aggressive human
selection of PE-multimer labeled cells and consecu-
and potentially protective immune cells that can
responses against a broad repertoire of MM-asso-
skin cancer that typically affects elderly and immu-
tive in-vitro expansion. We investigated MHC-mul-
be therapeutically reactivated. Therefore, the iden-
ciated antigens were detected and the TC frequen-
nosuppressed individuals. Recently, the Merkel Cell
timer enriched cultures from 29 MCC patients and
tification of major target antigens of spontaneous
cies were significantly higher compared to healthy
Polyomavirus (MCV) was found integrated into the
30 healthy donors. Interestingly, T-cell responses
tumor-reactive TC responses might be critical
donors. Interestingly, spontaneous TC frequencies
genome of 80% of MCC, and shown to play a role
against MCV derived peptides were seen more fre-
for the optimization of cancer immunotherapies.
and the number of TAA-reactive TC responses in-
in the oncogenic transformation. Polyomaviruses are
quent in the patients group than the healthy donor
Moreover, tumor antigen-specific antibody (Ab) re-
creased after tumor resection over time. Functional-
small circular DNA viruses encoding a T antigen on-
group (p<0.01). Furthermore it was evident that
sponses are often used as surrogate parameters of
reactive tumor-specific Treg were detected in some
coprotein locus. MCV expresses the small T and large
only the patient group showed responses against
tumor antigen-specific TC responses. However, a
patients, but in the majority of non-metastasized
T antigen together with the structural proteins VP1-3.
Large and Small T antigens, indicating that tar-
direct correlation of TC and Ab responses has so
MM patients an important role of Treg in controlling
Specific for the MCV genome integrated in the cancer
geting these antigens would specifically affect the
far not been systematically conducted.
spontaneous TAA-reactive TC responses could not
cells is a deletion of the large T antigen that renders
malignanT-cells.
Here, we examined the presence of spontaneous-
be identified. Moreover, spontaneous Ab responses
the virus non infectious. Immunotherapeutic target-
MCV specific T-cells has proven functionally effec-
ly induced tumor-reactive TCs and spontaneous
against the majority of analyzed TAAs were detect-
ing of virus positive tumors by CD8 T-cells could
tive in cytokine secretion and killing of peptide-
Ab responses against tumor-associated antigens
ed in the plasma of non-metastasized MM patients.
serve as an ideal strategy to treat MCC, however only
loaded targets cells, and we are currently investi-
(TAAs) in the peripheral blood of primary non-
Thereby, Ab responses were preferentially detected
one CD8 T-cell epitope (restricted to HLA-A24) was
gating the ability of the 26 potential T-cells epitopes
metastasized MM patients (stages 0, I, II) at dif-
against two or three TAAs in parallel per respond-
recently identified.
identified to induce MCC tumor cell killing, and
ferent time points after primary tumor resection.
ing MM patient and compared to healthy donors,
The aim of this study is to identify MCV encoded
thereby asses the therapeutic potential of these spe-
Furthermore, we investigated the effect of regula-
the number of Ab responses was generally elevated.
MHC class I restricted T-cell epitopes encoded by
cificities.
tory TC (Treg) depletion on TC reactivity against
But, in contrast to TC responses, the number of TAA-
small T, VP1 and the truncated sequence of large
TAAs in the absence of continuous stimulation by
specific Ab responses did not increase after tumor
T, by use of a high-throughput platform for T-cell
the tumor.
resection over time and in general, TAA-specific
epitope mapping. HLA ligands from the target
To analyze the presence and frequencies of spon-
Ab responses did not correlate with corresponding
protein was identified first by in silico ligand pre-
taneously induced preexisting tumor antigen-re-
spontaneous TC responses.
diction using the NetMHC and SYFPEITHI databas-
active TCs before and after depletion of Treg we
Summarized, our data show that spontaneous
es, and verified by experimental binding analyses
performed ex vivo short-term IFNγ ELISPOT assays.
tumor antigen-specific TC and Ab responses against
(MHC ELISA). A total of 236 peptides was identify
As antigen presenting cells, we used autologous
a broad repertoire of MM-associated antigens are
as ligands to HLA-A1, A2, A3, A11 and B7, and se-
dendritic cells which were pulsed with 50 amino
detectable in primary MM patients. However, the
lected for immunological analyses.
acid long synthetic polypeptides, spanning several
majority of patients so far examined showed no
We analyzed for the presence of MCV-specific T-cell
MHC class I- and II-restricted immunogenic regions
correlation of spontaneous TAA-specific TC and
responses in peripheral blood of MCC patients and
from 13 different TAAs (Melan-A/MART-1, NA17-A,
Ab responses.
+
146
+
147
096 | Immunomonitoring
097 | Immunomonitoring
Dissection of anti-CTLA4-induced cytotoxic T-cell responses in
melanoma
Tracking T-cell clones using high-throughput sequencing of antigen receptor CDR3 chains
1
1
1
1
2
Pia Kvistborg , Daisy Philips , Sander Kelderman , Bianca Heemskerk , Christian Ottensmeier ,
3
4
1
1
1
Antoni Ribas , Daniel Speiser , Christian Blank , John Haanen , Ton Schumacher .
1
Department of Immunology, Netherlands Cancer Institute, Amsterdam, Netherlands
2
Department of Medical Oncology, Southampton University Hospitals, Southampton, United
Kingdom
3
Department of Medicine, Jonsson Comprehensive Cancer Center, Los Angeles, US
4
Clinical Tumor Biology & Immunotherapy Group, Ludwig Center, Lausanne, Switzerland
148
1
2
1
1
Cindy Desmarais , Jessica Matthis , Robert Livinston , Ryan Emerson , Nishanth
1
1
3
2
3
Mathandan­ , Anna Sherwood , Jessica Andriesen , Helena Reijonen , Christopher Carlson ,
2
3
3
3
Gerold Nepom , Cassian Yee , Karan Cerosaletti , Harlan Robins 1
Adaptive Biotechnologies, Seattle, WA, U.S.A.
2
Benaroya Research Institute, Seattle, WA, U.S.A.
3
Fred Hutchinson Cancer Research Center, Seattle, WA, U.S.A.
There is strong evidence that melanoma-reactive
Interestingly, the magnitude of melanoma-specific
The cellular adaptive immune system generates a
clones). We used these samples to test the preci-
T cell responses induced by immunotherapeutic inter-
T-cell responses that was already detected prior to
remarkable breadth of diversity in Y-like antigen-
sion, accuracy, and sensitivity of our assay. We
ventions such as anti-CTLA4 (Ipilimumab) treatment
start of therapy was not significantly altered.
specific T-cell receptors (TCR) by combinatorial
found that TCRβ repertoire sequencing accurately
can exert clinically meaningful effects. However, at
These results establish the pattern of melanoma-
shuffling of gene segments in somatic cells. The
captures the frequencies of clones across five orders
present we have very little information on how these
specific T-cell reactivity induced by anti-CTLA4
existence of multiple V, D, and J gene segments in
of magnitude and is sensitive down to 1 in 100,000
therapies influence tumor-specific T-cell responses.
treatment and form a benchmark for evaluation of
the TCR loci (TCRβ/TCRα and TCRδ/TCRγ) permits
cells. These results indicate that T-cell receptor
Furthermore, as the number of potential melanoma-
other immunotherapeutic interventions, like anti-
large combinatorial diversity in receptor composi-
sequencing is an accurate method to track T-cell
associated antigens to which these responses can be
PD1 treatment, that are currently undergoing clini-
tion, and template-independent deletion or inser-
clones of interest. Potential applications include
directed is very high, classical strategies to map cy-
cal evaluation. Furthermore, the data suggest that
tion of nucleotides at the V-J, V-D, and D-J junctions
tracking Minimal Residual Disease and tracking
totoxic T-cell reactivity do not suffice. Knowledge of
the clinical activity of Ipilimumab may be mostly
further adds to the potential diversity. Because the
clones used for Adoptive Immune Therapy.
such reactivities would be useful to design targeted
due to epitope spreading, rather than through en-
potential diversity of receptors is large (approxi-
strategies, selectively aiming to induce immune re-
hancement of pre-existing immune activity.
mately 10 million different TCRβ), it is improbable
activity against these antigens.
to randomly converge on the same CDR3 sequence,
To address this issue, we have recently developed
effectively making each CDR3 sequence a unique
MHC multimer-based technology allowing high-
nucleotide tag for a T-cell clone. However, the diver-
throughput dissection of therapy-induced CTL im-
sity of possible receptors is huge, making identify-
munity. We have now used this platform to monitor
ing and tracking clones difficult.
immune reactivity against a panel of 145 melano-
We have developed immunoSEQ, a method that
ma-associated epitopes in patients receiving Ipili-
amplifies rearranged TCRβ CDR3 sequences and
mumab treatment.
uses high throughput sequencing to sequence mil-
Comparison of PBMC samples from 31 melanoma
lions of chains per sample. This technology permits
patients pre- and post-therapy demonstrates a sig-
“clone-tracking” by both verifying the presence/
nificant increase in the number of detectable mel-
absence of specific T-cell clones and estimating the
anoma-associated CD8 T-cell responses (p=0.02).
frequency of these clones within the overall rep-
Furthermore, kinetic data on T-cell responses
ertoire. We verified the feasibility of our assay to
during Ipilimumab therapy suggest that this
quantitatively track clones of interest by sequencing
broadening generally occurs within weeks after
the repertoire of samples doped with T-cell clones
start of therapy. The pattern of reactivities detect-
across a 5-fold range (10-1,000,000 cells). Two in-
ed is highly patient specific, and this is most pro-
dependent laboratories blindly sent us samples
nounced for reactivities directed against cancer/
with clones spiked into a diverse background at
testis antigens.
3 concentrations (4 clones) or 4 concentrations (2
149
098 | Immunomonitoring
099 | Immunomonitoring
HLA subtype variation strongly affects MHC multimer-based
monitoring of antigen specific CD8 T-cell responses
Cancer vaccination with telomerase peptide GV1001: The immune response relates to clinical outcome
1
1
1
1
1, 2, 3
2, 4
3
2
2
Marit van Buuren , Feline Dijkgraaf , Carsten Linnemann , Pia Kvistborg ,
1
2
2
1
Mireille Toebes , Cynthia Chang , Gijsbert Grotenbreg and Ton Schumacher Jon Amund Kyte
, Steinar Aamdal , Else Marit Suso , Paal Brunsvig , Svein Dueland ,
3
5
6
3, 4
Gaute L Hansen , Christian Kersten , Stein Sundstrøm and Gustav Gaudernack 1
Department of Immunology, Netherlands Cancer Institute, Amsterdam, Netherlands
1
Section for Cell Therapy, Dept. of Oncology, Oslo University Hospital, Oslo, Norway
Department of Microbiology, National University of Singapore, Singapore
2
Section for Clinical Cancer Research, Dept. of Oncology, Oslo University Hospital
3
Section for Immunology, Cancer Research Institute, Oslo University Hospital
4
Faculty of Medicine, University of Oslo
5
Center of Cancer Treatment, Southern Hospital of Norway, Kristiansand, Norway
6
Department of oncology, St Olav’s Hospital, Trondheim, Norway
2
150
In the field of immunomonitoring, the use of
subtypes, as performed here for HLA-A*02, will
The human telomerase reverse transcriptase
in 13 subjects. Immune responders recorded a
Major Histocompatibility Complex (MHC) multim-
therefore be of importance for the development of
(hTERT) is overexpressed in most human cancers.
median progression-free survival (PFS) of 371 days,
ers coupled to fluorochromes is a widely applied
personalized immunomonitoring.
We have developed the cancer vaccine GV1001, a
compared to 182 days for non-responders (p=0.20).
method to monitor antigen specific T-cell responses
16-mer hTERT peptide. Our previous trials with
We attempted to investigate why some immune
in patients using flow cytometry. T-cells recognize
GV1001 as monotherapy demonstrated immune
responders had apparent clinical benefit, while
their peptide in a MHC restricted manner and it
responses in ~60% of lung- or pancreatic cancer
others did not. Interestingly, patients developing
is therefore of obvious importance to know the
patients. Here, we summarize main findings in two
long term T-cell memory survived longer than those
Human Leukocyte Antigen (HLA) haplotypes of
recent trials combing GV1001 with chemo/radio-
rapidly losing their responses. Contrary to subjects
patients. Such HLA typing is often performed by
therapy and report results from immunological
without clinical response, long term survivors
antibody staining or low resolution genotyping,
follow-up studies.
developed T-cell responses against a spectrum of
which gives (2-digit) information about the HLA
In a proof-of-principle trial in 25 stage IV mela-
hTERT peptides unrelated to the GV1001 sequence.
types expressed by an individual but ignores any
noma patients (NCT01247623), we evaluated com-
The immune responses in long term survivors also
(4-digit) subtype variation.
bined therapy with temozolomide and GV1001.
exhibited other characteristics of possible clinical
In this project we set out to assess to what extent
The treatment was well tolerated, and a GV1001-
significance, including high IFNγ/IL-10 ratios, pol-
the small sequence changes that exist between HLA
specific immune response was demonstrated in
yfunctional cytokine profiles, combined T-helper/
subtypes affect the ability to detect antigen-specific
18/23 evaluated subjects. Five patients developed
cytotoxic responses and recognition of naturally
T-cells for a given peptide. To this purpose, we first
partial tumor regression and six more recorded
processed antigens. Finally, immune responders
developed MHC peptide exchange technology for
stable disease. The clinical responses developed
with a low percentage of CD4 CD25 Foxp3 T-cells
a series of eight different HLA A*02 subtypes. We
gradually over years, contrary to what is expected
had extended PFS, while no correlation between
then created HLA multimers for each of these HLA
from chemotherapy. Survival compared favourably
myeloid suppressor cell counts and PFS was ob-
A*02 subtypes with 10 different peptide antigens
to matched controls from a benchmark meta-anal-
served.
for which we had created HLA A*02:01 restricted
ysis (one year: 44% versus 24%, two years: 16%
The immune response rates in the trials summa-
T-cell clones. Importantly, while this set of T-cell
versus 6.6%).
rized above were considerable compared to pre-
clones was reliably stained with the matched HLA
A phase II trial (NCT00509457) was conducted in
vious trials with GV1001 as monotherapy, while
A*0201 multimer, staining with multimers for other
23 patients with inoperable stage III non-small cell
low toxicity was retained. Our results support the
HLA A*02 subtypes was highly variable.
lung cancer (NSCLC). The patients received radio-
concept of combining chemo/radiotherapy with
We conclude that even minor sequence variation
therapy and weekly docetaxel, followed by GV1001
vaccination and warrant further studies of GV1001.
between HLA subtypes can greatly influence the
vaccination. We observed no serious adverse
In cooperation with Kael Gemvax, we have initi-
ability to detect antigen-specific T-cell responses
events. A GV1001-specific immune response devel-
ated a randomized GV1001 trial in stage III NSCLC
by MHC multimer staining. Generation of MHC-
oped in 16/20 evaluable patients. Long term immu-
patients.
based monitoring technology for large sets of HLA
no-monitoring demonstrated persisting responses
+
+
+
151
100 | Immunomonitoring
101 | Immunomonitoring
NY-ESO-1 and Melan-A-reactive T-cells are predictive for the
clinical outcome of late-stage melanoma patients
Reference samples to control performance of HLA-peptide multimer staining experiments – First results from a proficiency
panel testing
1
2
1
2
1, 2
1, 2
3
2
1, 2
4
Henning Zelba , Benjamin Weide , Evelyna Derhovanessian , Claus Garbe , Graham
1
Pawelec N. Bidmon , H. Filbert , C. Gouttefangeas , U. Sahin , S. Attig , S. H. van der Burg ,
2
C.M. Britten 1
Department of Internal Medicine II, Section for Transplantation Immunology and Immuno­
haematology, University Hospital Tübingen, Germany
1
III. Medical Department, University Medical Center of the Johannes Gutenberg University
Mainz, Mainz, Germany
2
Department of Dermatology, Division of Dermatooncology, University Hospital Tübingen, Germany
2
Translational Oncology at the University Medical Center of the Johannes Gutenberg University
Mainz (TRON), Mainz, Germany
3
Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen,
Tübingen, Germany
4
Department of Clinical Oncology, Leiden University Medical Center, Leiden, Netherlands
For the CIMT Immunoguiding program
Chemotherapy is still the standard treatment for
PBMCs, the assay readout was an intracellular cy-
The lack of standard control reagents for immu-
Results from this proficiency panel show (i) fea-
late-stage melanoma patients, but clinical outcome
tokine staining using multicolor flow cytometry
nological T-cell assays prohibits comparability of
sibility of RS use across 12 participating labs, (ii)
is poor. Alternative approaches are required and
to detect six cytokines simultaneously (IFN-γ,
results generated in one lab over time and across in-
high reproducibility of results generated between
immunotherapy is becoming increasingly ex-
TNF, IL-2, IL-4, IL-17 and IL-10). This allowed us
stitutions. Therefore commonly available reference
two time points, (iii) low background staining
ploited for melanoma management. Clinical trials
to analyze the phenotype and the function of the
samples (RS) that can be manufactured at large
of relevant dextramers to the negative control RS
of vaccines and immunomodulators are yielding
T-cell antigen-response at the single-cell level.
scale and continuously lead to stable results are ur-
batch, (iv) lack of cross-reactivity of the irrelevant
impressive results, but still only in a minority of
We show here that the presence of NY-ESO-1 and/
gently needed. We established a process to generate
multimer with the RS and (v) compatibility of RS
patients. Patient parameters associated with suc-
or Melan-A-reactive T-cells was significantly as-
stable standard samples to control the performance
to use with multiple sources of HLA-A2-peptide
cessful therapy are largely undefined, and whether
sociated with survival and predicted the clinical
of HLA-peptide multimer experiments and other
multimers.
targeting particular antigens can result in a better
outcome even more strongly than the M category or
T-cell assays. In our hands, these RS showed high
In summary the results from this proficiency panel
clinical outcome than others is not clear. Further-
type of therapy received. NY-ESO-1 was generally
reproducibility and stability. The purpose of this
suggest that RS are useful tools to control immune
more, predictive markers are lacking. Lactate de-
recognized more frequently by CD4 than by CD8
+
exploratory proficiency panel (CIP_ID12_MUL/E)
assay performance over time or across institutions.
hydrogenase is thus far the only well-accepted bio-
T-cells, both being associated with a positive effect
was to find out how RS perform in the hands of
marker for malignant melanoma. The amount of
on patient survival. In contrast, Melan-A mainly
experienced investigators in a heterogeneous group
List of participants:
+
+
+
LDH (and other markers), however, does not allow
stimulated CD8 T-cells and recognition by CD4
of labs that apply different assay protocols.
1) A. Cazaly – Southampton General Hospital, England
the prediction of the clinical outcome of a single
T-cells was associated with a bad clinical outcome.
PBMCs from a HLA-A2 positive buffy coat donor
2) C. Gouttefangeas – University of Tübingen, Germany
patient. Initially, we observed in a small cohort
Taken together, our data confirm the important role
were used to generate three different RS batches
3) M. Hasan – Institut Pasteur, Paris, France
of late-stage melanoma patients that the presence
of NY-ESO-1 and Melan-A as target antigens of first
either lacking (negative control) or containing high
4) T. Jakobsen – Immudex, Copenhagen, Denmark
in the peripheral blood of antigen-reactive T-cells
choice for melanoma-immunotherapy. The fact that
or low numbers of CD8 T-cells specific for a HLA-A2
recognizing the cancer/testis antigen NY-ESO-1 in
each antigen is preferentially detected by a certain
restricted peptide derived from the tumor antigen
5) P. Kvistborg – Netherlands Cancer Institute,
Amsterdam, Netherlands
vitro seems to correlate with survival. In order to
cell type and that this had strong impact on sur-
NY-ESO-1. Quality-controlled aliquots of the RS
determine whether the presence of T-cells capable
vival, at least for Melan-A, should be considered
batches were shipped to 12 participating labs to-
of detecting tumor-associated antigens allows pre-
for upcoming trials.
gether with an irrelevant HLA-A2 and a specified
7) R. Mendrzyk – Immatics biotechnologies GmbH,
Tübingen, Germany
diction of clinical outcome, we assessed T-cell re-
NY-ESO-1 HLA-A2 dextramers. Each laboratory was
8) P. Palluch – Helmholtz-Zentrum München, Germany
sponses against 4 common melanoma-associated
asked to use the three RS batches in HLA- peptide
9) S. Reker Hadrup – University Hospital Herlev, Denmark
antigens in 116 stage IV melanoma patients.
multimer staining experiments at two independ-
We assayed functional antigen-reactive T-cells
ent time points using the two centrally provided
10)M.J.P. Schoenmaekers-Welters – Leiden University Medical Center, Netherlands
recognizing NY-ESO-1, Melan-A, MAGE-A3 and
dextramers. Optionally, participants were allowed
survivin after 12 days of in vitro expansion with
to perform a third data set using a locally available
overlapping peptides representing these molecules.
NY-ESO-specific HLA-A2-peptide multimer.
6) J. Matsuzaki – Roswell Park Cancer Institute, Buffalo, United States
11)J. de Vries – Radboud University Nijmegen Medical Centre, Netherlands
12)N. Bidmon – University Medical Center of the Johannes Gutenberg University, Mainz, Germany
After restimulation with autologous antigen-pulsed
152
153
102 | Immunomonitoring
103 | Immunomonitoring
Reference Samples as stable controls for T-cell assays
Treg Flow Cytometry Staining – Hunting a FOX
1, 2
1
2
2
2
1, 2
2
N. Bidmon , S. Attig , T. Omokoko , P. Simon , S. Kreiter , H. Filbert , R. Rae ,
2
3
2
U. Sahin , S. H. van der Burg , C.M. Britten 1
III. Medical Department, University Medical Center of the Johannes Gutenberg University
Mainz, Mainz, Germany
2
Translational Oncology at the University Medical Center of the Johannes Gutenberg University
Mainz (TRON), Mainz, Germany
3
Department of Clinical Oncology, Leiden University Medical Center, Leiden, Netherlands
154
1
2
3
1
1
Richard Rae , Sebastian Attig , Cecile Gouttefangeas , Ugur Sahin and Cedrik Britten 1
TRON - Translationale Onkologie an der Universitätsmedizin der Johannes Gutenberg-Universität Mainz gGmbH
2
Universitätsmedizin der Johannes Gutenberg-Universität III. Med. Klinik / TVZ
3
Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen,
Germany
Different assay platforms are used for the quan-
ing a high reproducibility of results. Long-term
When enumerating Tregs using flow cytometry,
primarily capture the very same Tregs as a panel
tification of antigen-specific T-cell responses on
studies confirmed the stability of the RS for over
there is a large selection of markers available for
that requires the fixation and permeabilisation of
a single cell level. Reference Samples (RS) that
6 months. For ICS experiments we generated and
use. CD3 and CD4 are essential to find the CD4
the nucleus to allow FoxP3 staining. This Simple
contain a defined number of antigen-specific
tested an HLA class I (B7-restricted) and an HLA
T-cells – the parent population including the
staining and gating also has the added advantage
T-cells to continuously provide stable signals in
class II (DBR1*0401-restricted) specific RS and
Tregs – but truly Treg specific markers are not so
of excluding CD25 Foxp3 T-cells that have been
repetitively performed assays are urgently needed.
confirmed high reproducibility of results (CV~
easy to select. We have designed and optimised
shown to only transiently express FoxP3 and to be
We developed an optimized process to generate
20%) generated from subsequently tested aliquots
a flow panel containing the Treg markers CD127,
not suppressive. Finally, a strategy to capture Tregs
RS containing a defined number of T-cell recep-
for four commonly used T-cell activation markers.
CD25, FoxP3, CD103 and CCR4 along with 3 differ-
that is solely based on staining of surface markers
tor (TCR)-engineered lymphocytes with maximum
The RS showed high stability even after 6 month of
ent gating strategies using combinations of these
allows for sorting of viable cells for use in func-
functionality and high viability. Applying the
storage in liquid nitrogen. We also generated prom-
markers. Surprisingly, we have found that the cells
tional assays.
-
+
+
optimized process, multiple RS batches contain-
ising results in ELISPOT assays using multiple RS
that fall in the “Classical” Treg gating (CD4 CD-
ing defined concentrations of antigen-specific T
batches (B7- and DRB1*0401-restricted) containing
127
lymphocytes for two known tumor antigens (ty-
defined numbers of NY-ESO-1 specific T-cells.
high concordance to those found using a “Simple”
rosinase and NY-ESO-1) were generated. At CIMT
In summary we were able to establish the proof of
(CD4
2011 we presented the results of the production and
concept for three major assay platforms using an op-
strategy, namely a mean of ~80% concordance of
optimization process for HLA-A2-restricted tyrosi-
timized, newly developed manufacturing process
events in 10 healthy donors. Moreover, quantifi-
nase and NY-ESO-1 specific RS that were applied
for standard controls. We conclude that optimized
cation of Tregs using the “Simple” gating strategy
in HLA-peptide multimer staining experiments. In
TCR RNA-engineered RS can serve as a stable and
gives highly reproducible results with an intra- and
this study we (i) show extended stability data for
defined source of antigen-specific T-cells that exert
inter-assay coefficient of variance below 7%.
RS in HLA-peptide multimer experiments, (ii) the
full functionality. Multiple aliquots of such RS can
We have also shown that additional gating with
performance characteristics and stability data for
be used repetitively to facilitate assay development
CCR4 (“Alternative gating”), that has recently been
HLA-B7- and DRB1*0401-restricted RS applied in
and to serve as internal controls in immunological
proposed by flow cytometry networks in the USA,
intracellular cytokine staining (ICS) experiments
assays applied in correlative biomarker studies.
is not beneficial in our hands to help identifying
low
+
hi
+
CD25 FoxP3 T lymphocytes) have a very
low
CD127
CD25
hi
T lymphocytes) gating
and (iii) first proof of concept experiments for RS
Tregs. These findings have also been confirmed in
technology used in ELISPOT assays.
cancer patient peripheral blood samples as well as
Single aliquots from generated HLA class I (A2-
tumour infiltrating lymphocyte samples. We have
restricted) specific RS were thawed and indepen-
also shown that CD103 is highly expressed in TIL
dently tested in multimer stainings using HLA-
samples but is not highly expressed on the Tregs
peptide tetramers or dextramers. The coefficient
of the TILs.
of variation for the thawed RS in the multimer
We propose that in future flow panels only extra-
staining experiments was less than 10% indicat-
cellular Treg markers should be stained as this will
155
104 | Immunomonitoring
105 | Immunomonitoring
Activation, Dysfunction and Retention of Antigen-Reactive
T-cells in the Vaccine Site Microenvironment after Multipeptide
Vaccine in Incomplete Freund’s Adjuvant
Myeloid sub-populations are correlated to survival in patients
with cervical cancer
1, 2
1
1
1
1, 2
2
2
3
Sofia M. Shea , Walter C. Olson , Chantel McSkimming , Elise P. Salerno ,
1
1, 3
1
1
Donna H. Deacon , Jochen T. Schaefer , Kim Bullock , Craig L. Slingluff Jr. Peggy de Vos van Steenwijk , Tamara Ramwadhoebee , Eline Doorduijn , Arko Gorter ,
1
2
3
Mariëtte van Poelgeest , Sjoerd van der Burg , Ekaterina Jordanova 1
Division of Surgical Oncology, Department of Surgery, University of Virginia, Charlottesville, VA USA
1
Departement of Gynecology, Leiden University Medical Center, Leiden, Netherlands
Department of Dermatology, University of Virginia, Charlottesville, VA USA
2
Departement of Clinical Oncology, Leiden University Medical Center, Leiden, Netherlands
Department of Dermatology, University of Connecticut, Farmington, CT USA
3
Departement of Pathology, Leiden University Medical Center, Leiden, Netherlands
2
3
Melanoma vaccines have been associated with regres-
an early Th2 dominant microenvironment with subse-
sions in 3-6% of patients with advanced measurable
quent accumulation of eosinophils.
disease, but have not been optimized. While defined
By flow cytometry, we found that the majority of CD8
peptides are attractive vaccine candidates; the admin-
T-cells (62-65%) in the VSME were effector memory
istration of these peptides in water-in-oil emulsions of
cells (CCR7 /CD45RA ). The vaccine sites for group
incomplete Freund adjuvants, may limit immunogenic-
2 were enriched for CD8 T-cells expressing T-cell re-
ity and clinical benefit. We hypothesized that lymphoid
ceptors for peptides in the vaccine (tetramer-positive
neogenesis may be induced at sites of repeated injection
T-cells) compared to the blood (median 1.42% vs.
with cancer vaccines, and that this may have both posi-
0.25%, respectively, p< 0.01, t-test) and were low in
tive and negative impacts on vaccine efficacy.
replicate vaccine sites after IFA only (median 0.34%).
We conducted a randomized clinical trial in 44 patients
Among tetramer positive cells in the vaccine site, 86%
focused on characterization of cellular and molecular
were activated (CD69+) and most of these cells (57%)
events, over time, induced at sites of immunization
expressed CXCR3. However, only 5% of the tetram-
with peptides in an incomplete Freund’s adjuvant,
er+ CD8 T-cells produced interferon γ by ELIspot after
Montanide ISA-51 (IFA). Patients with resected AJCC
stimulation with peptides in the vaccines. Thus, anti-
stage IIB-IV melanoma, expressing HLA-A1, A2, A3,
gen-reactive effector memory CD8 T-cells accumulated
or A11, were injected in two skin sites. At the primary
at sites of vaccination with peptides plus IFA, but were
vaccine site, the full vaccine (12 Class I MHC restricted
either exhausted or in a refractory state due to pro-
melanoma peptides and a tetanus helper peptide emul-
longed antigen exposure. The accumulation of these
sified in IFA) was administered to all participants at
T-cells in the VSME raises questions about whether
days 1, 8, 15, 29, 36, and 43. At a replicate vaccine site,
they are being induced there or are trafficking after
the injectate differed between the 2 primary arms of
activation in vaccine-draining nodes. Antigen-specific
the study: participants in study group 1 received IFA
T-cells in the VSME for group 2 expressed retention
only; those in study group 2 received the full vaccine.
integrins αEβ7 and α1β1 at high frequency (38 and
Within each study group, participants were further
77% respectively); whereas both are almost complete-
randomized to undergo excisional biopsy of the rep-
ly absent from circulating antigen-non-reactive T-cells
licate site on one of five possible days; day 1, 8, 22,
(3% and 5% respectively).
50, or 85.
Thus, although IFA is a useful adjuvant in peptide
Histologic analysis of the vaccine site microenvironment
vaccines by generating circulating antigen reactive
+
neg
(VSME) revealed a T-cell (CD3 ) predominance. Both
T-cells, the cytokine milieu generated by it is subopti-
the T-cell and B-cell components expanded with repeat
mal. There is evidence that the combination of IFA and
vaccinations; however, the T-cell component expanded
antigen leads to retention of antigen-specific T-cells at
+
more quickly. Mature (CD83 ) dendritic cells were found
+
the VSME; and therefore the cells may not be able to
in the papillary dermis, and immature (CD1a ) dendritic
exit the VSME to provide systemic immunity. Further
cells were found within the inflammatory infiltrates, but
characterization of the VSME is warranted to guide
+
neither expanded significantly. FoxP3 cells increased
+
after 3 vaccinations. Analysis of the CD4 cells showed
156
neg
selection of optimal adjuvants for use with peptide
vaccines.
Introduction: Cancer development is a proces
the CD14 single positive cells in the tumor corre-
defined by the tumor within its micro-environment
lates to a beter survival (p=0.011), negative lym-
which is influenced by the host immune system.
phnodes (p=0.006) and no parametrial invasion
Various variables have been correlated to survival
(0.046). The only myeloid celltype that seems to
in cervical cancer, yet little is known about the
have a tumor promoting effect is the CD163 single
presence and role of myeloid subsets.
positive cell in the stroma, which was correlated to
Aim: The aim of this study was to investigate the
positive lymphnodestatus (0.037), a dieper tumor
presents of myeloid cells in a group of 87 cervical
infiltration (0.027) and to a worse suvival (though
carcinoma patients undergoing an hyseterctomy and
this was not significant p=0.077).
their correlation to clinical parameters and survival.
Conclusion: CD14 myeloid cells in the tumor epi-
Materials and methods: Paraffin-embedded tissue
thelium have a positive effect on survival, whereas
sections from 87 patients were stained by immuno-
CD163 single positive cells in the stroma are cor-
histochemistry using antibodies to CD33, CD14 and
related to worse survival.
+
CD163 and counted manually. Statistical analysis
was performed using the Chi-squared test. Kaplan–
Meier survival curves were generated to assess differences in cumulative overall survival.
Results: The most commen myeloid cell types in the
+
tumor epithelium were CD33 polymorphonuclear
+
+
cells (31%), CD14 CD33 CD163 triple positive cells
(19%) and CD14 single positive cells (12%). The cell
distribution in the stroma was similar yet differed
slightly with more CD163 single positive cells (10%)
+
following the most abundant CD33 polymorpho+
+
nuclear cells (39%) and CD14 CD33 CD163 triple
+
positive cells (18%). The presence of CD14 myeloid
cells in the tumor epithelium has an independent
positive effect on survival (p= 0.03). Expecially
157
106 | Immunomonitoring
107 | Immunomonitoring
Quantitative determination of tumor-specific Treg in lymphatic
compartments of patients
IMA910, a novel multi-peptide cancer vaccine for advanced
colorectal cancer, induces multiple CD8+ and CD4+ T-cell
­responses associated with improved survival
1
1
1
1
1
Hans-Henning Schmidt , Felix Jerg Hartmann , Yingzi Ge , Philipp Beckhove 1
Department of Translational Immunology, DKFZ, Heidelberg, Germany
1
1
1
1
immatics biotechnologies, Tübingen, Germany
2
Department of Oncology, Hematology, Immunology, Rheumatology and Pulmonology,
University of Tübingen, Tübingen, Germany
3
Department of Internal Medicine and Chemotherapy “B”, National Institute of Oncology,
­Budapest, Hungary
4
Department of Clinical and Experimental Oncology, Centrum Onkologii Instytut im.
Marii Skłodowskiej-Curie, Gliwice, Poland
5
Department of Oncotherapy, University of Szeged, Szeged, Hungary
6
Department of Pathology and Diagnostics, University of Verona, Verona, Italy
Regulatory T-cells (Treg) are a subset of CD4
+
Treg specificity. By this method peptides derived
T-cells which are able to suppress immune reac-
from Mammaglobin A and Her-2 specifically rec-
IMA910 is a novel peptide-based vaccine consisting
multiple CD8 and CD4 T-cell responses, respective-
tions. In tumor immunology Treg are known to
ognized by Treg were identified in up to 35% of
of 10 HLA-A*02 and 3 HLA-DR binding synthetic pep-
ly. Patients that received imiquimod presented with
play a detrimental role as elevated tumor-infiltrat-
breast cancer patients. HLA tetramers were formed
tides that were identified based on natural presenta-
significantly more multiple CD8 cell responses as
ing Treg correlate with poor prognosis in several
with the defined antigens. With these the spatial
tion on human colorectal cancer (CRC) samples.
detected by ICS (p=0.016) and a trend to increased
cancer types. It has been shown that engagement of
distribution between peripheral blood and bone
IMA910 was characterized in a phase I/II trial in
response magnitudes by HLA-multimer anaysis
cognate antigen presented by suitable human leu-
marrow and the phenotype of TAA specific T-cells
advanced/metastatic CRC patients with stable or re-
(p=0.12).
kocyte antigen (HLA) molecules can activate Treg
in patients was examined.
sponding disease after 12 weeks of first-line oxalipl-
IMA910 antigen-specific multiple CD8 and multi-
suppressive activity. However, antigen specificity
Tumor specific T-cells could be found in the major-
atin-based therapy (92 HLA-A*02 patients in total).
ple CD4
of cancer patient derived Treg is poorly defined.
ity of analyzed patients with an average frequency
After immunomodulation with a single dose of cyclo-
with significantly better clinical outcome. The as-
HLA tetramers can be used to stain T-cells specific
of 0.2% of the CD4 TC. In previous experiments
phosphamide (300 mg/m ), patients were immunized
sociation was most pronounced for patients with
for the peptide they recognize. For the generation of
we could show that the frequency of Treg in the
intradermally (up to 16 vaccinations) with IMA910 in
both multiple CD8 and multiple CD4 responses.
HLA tetramers the sequence of the peptide and the
bone marrow of patients is significantly reduced
combination with GM-CSF without (cohort 1; n=66)
These patients had significantly higher DCR at 6
HLA it is presented on have to be known.
compared to the peripheral blood. With the tetram-
or with (cohort 2; n=26) topically applied imiquimod.
months (p=0.002), improved TTP (p=0.006) and
Our previous studies showed that an otherwise
ers we could prove that the same tendency is true
Before and post vaccination, patients were analyzed
improved PFS (p=0.009) than other patients. Most
latent T effector/memory response against some
for tumor specific Treg. In addition we found that
by HLA-multimer assay and intra-cellular cytokine
importantly, a trend for prolonged OS was also
tumor-associated antigens (TAAs) was enabled by
the frequency of tumor specific Treg of the total
(ICS) assay for CD8
the depletion of Treg. This was especially true for
Treg is significantly lower in the bone marrow than
assay for CD4 T-cell responses. As immune status
0.53).
two TAAs in breast cancer Mammaglobin A and
in the blood of breast cancer patients.
biomarkers, 6 phenotypically defined myeloid derived
In the study population, levels of 5 different MDSC
Her-2 showing the presence of Treg with respective
To our knowledge this is the first time that tetram-
suppressor cell populations (MDSC1-6) were analyzed
phenotypes were significantly increased as compared
specificities in breast cancer patients. Polypeptides
ers were used to stain tumor specific regulatory
prior to immunotherapy. Tumor status of patients was
to age/gender matched controls. High MDSC levels
(50 amino acids) of these antigens recognized by
T-cells in the blood and the bone marrow. With
monitored repeatedly by CT/MRI according to RECIST
were associated with fewer immune responses and for
Treg were identified in a Treg-specificity assay.
this method we could analyze the localization of
and corresponding tumor scans were reviewed cen-
MDSC4 and MDSC5 high frequencies were associated
The bioinformatics tool SYFPEITHI was used to
tumor specific Treg in lymphatic compartments of
trally. Clinical assessment included disease control
with shorter OS (p=0.007 and p=0.019, respectively).
predict potential HLA class II presented peptides of
breast cancer patients. The results will give a better
rate (DCR), time to progression (TTP), progression-
Interestingly, the same MDSC phenotypes had been
these polypeptides. These were tested for their spe-
understanding of Treg immunity, which might lead
free survival (PFS) and overall survival (OS).
previously observed to be associated with survival in
cific recognition by Treg in HLA-DR typed breast
to the improvement of therapies.
IMA910 was immunogenic in 73/81 (90%) evaluable
renal cell carcinoma patients implying a more general
patients, with 43% and 65% of patients mounting
role for these populations.
cancer patients by the same functional assay of
158
1
Steffen Walter , Sabrina Kuttruff , Sarah Kutscher , Andrea Mayer , Oliver Schoor ,
1
2
3
4
5
Jörg Ludwig , Frank Mayer , Erika Hitre , Elżbieta Nowara , László Torday ,
1
1
1
1
6
Bernhard Rössler , Dominik Maurer , Verona Vass , Juha Lindner , Vincenzo Bronte ,
1
1
1
1
1
Nina Pawlowski , Claudia Trautwein , Jörn Dengjel , Norbert Hilf , Toni Weinschenk ,
1
1
1
­Jürgen Frisch , Carsten Reinhardt , Harpreet Singh +
+
2
+
T-cell responses and by ICS
+
+
+
+
+
+
responses were individually associated
+
+
observed in these patients (p=0.088, hazard ratio
159
108 | Immunomonitoring
109 | Immunomonitoring
Immune Monitoring of Poxvirus based Cancer Immunotherapies
Homing receptor expression by T-cells infiltrating the metas­tatic
melanoma microenvironment and relevance to combination
immune therapy
Rachel Owen, Fatema Legrand, Amanda Enstrom, Olivia Hwang, Gayatri Paranjpe, Jinsoo
Joo, Joy Su, Bernadette Callejo, Alex Chung, Jess Nolin, Olga Bandman, Wayne Godfrey,
Reiner Laus, Alain Delcayre
Elise P Salerno , Walter C Olson , Chantel McSkimming , Sofia M Shea , Craig L Slingluff, Jr Department of Research and Development, BN ImmunoTherapeutics Inc., Mountain View, CA, USA
160
1
1
1
1, 2
1
Department of Surgery, University of Virginia, Charlottesville, VA USA
2
Department of Dermatology, University of Virginia, Charlottesville, VA USA
1
T-cell infiltration into the metastatic melanoma micro-
in small bowel metastases (42 ± 21%), but not in other
environment (MME) correlates with improved patient
sites (8 ± 8%, p=0.001). Among antigen-experienced
survival and response to immune therapy. However,
CD8 T-cells, CCR5 cells were enriched (85 ± 11%)
diffuse T-cell infiltration is evident in less than 10%
in distant metastases, compared to TIN (61 ± 18%,
of melanoma metastases, and little is known about
p=0.014) and PBMC and LN (55 ± 7%, p=0.001).
mechanisms governing T-cell infiltration into human
Distant metastases had more CCR4 cells (58 ± 5%)
+
+
+
melanoma metastases, or about how infiltration may
than TIN (43 ± 16%, p=0.033), PBMC and LN (44 ±
BN ImmunoTherapeutics Inc. is developing cancer
tion assay. In addition, humoral responses were as-
be augmented therapeutically. This study was designed
11%, p=0.021). CXCR3 expression was low in metasta-
immunotherapies using poxvirus based vectors
sessed by ELISA. Immune monitoring revealed the
to identify homing receptors on T-cells infiltrating the
ses (14 ± 12%) compared to LN (26 ± 5%), and CLA+
that encode heterologous cancer antigens. The
induction of humoral and/or T-cell responses spe-
MME. We hypothesized that T-cells in the MME would
cells were underrepresented in metastases (10 ± 2%)
MVA-BN® vector is a highly attenuated vaccinia
cific to the transgene encoded by the vector in the
be enriched for homing receptors relevant to the tissue
compared to PBMC (15 ± 2%, NS). Among antigen-
virus that is non-replicating in humans and has
majority of patients treated with MVA-BN®-HER2
site, including cutaneous leukocyte antigen (CLA) for
experienced CD4 cells, CCR5 cells were markedly
been shown to be an immunogenic vaccine. This
and MVA-BN®-PRO. The presence of a pre-existing
skin metastases and chemokine receptor 9 (CCR9) for
increased in skin and bowel metastases (72 ± 15%)
vector has been utilized to express tumor specific
immune response to MVA did not impair the in-
small bowel metastases. Also, because CCR4, CCR5
compared to PBMC, LN and TIN (13 ± 7%, p<0.001),
proteins for breast cancer (MVA-BN®-HER2) and
duction of transgene specific immune responses.
and CXCR3 have been implicated in tumor homing,
and other findings were similar to those for CD8s. A
prostate (MVA-BN®-PRO) cancer. MVA-BN®-based
Additionally, vaccine-induced determinant spread-
we hypothesized that T-cells expressing them would
striking finding was a marked increase in expression of
immunotherapy vectors have been tested in proof-
ing was evident in tumor-bearing patients treated
be increased in the MME. Single cell suspensions from
retention integrins in CD8 T-cells: whereas αEβ7 was
of-concept animal models as well as in early stage
with MVA-BN®-HER2 and MVA-BN®-PRO. The
14 melanoma metastases representing three metastatic
expressed on a mean of only 3 ± 1% of CD8 T-cells
clinical settings.
therapies were well tolerated with no dose-limiting
sites: tumor-infiltrated lymph node (TIN, n=8), skin and
in PBMC and LN, this increased to 18%, 28%, and 76%
MVA-BN®-HER2 utilizes a poxviral vector that
toxicities or serious adverse events reported. The
subcutaneous tissue (skin, n=3) and small bowel (n=3)
in TIN, skin metastases, and small bowel metastases,
encodes a modified and truncated form of the
data suggest that MVA-BN®-HER2 and MVA-BN®-
were evaluated by multiparameter flow cytometry.
respectively (SDs 13-17%), One-way ANOVA p<0.001).
HER-2 epidermal growth factor receptor (HER-2
PRO are well-tolerated, immunogenic, and support
These were compared to two benign lymph nodes (LN)
There were similar increases in α1β1 and α2β1 retention
extracellular domain plus 2 tetanus toxoid peptide
going forward with larger efficacy trials.
and normal donor peripheral blood mononuclear cells
integrins in metastases.
epitopes), and has been tested in metastatic and ad-
(PBMC). Comparison of means was performed using
Thus, the phenotype of T-cells infiltrating melanoma me-
juvant breast cancer. MVA-BN®-PRO has been en-
2-tailed t-tests for independent samples.
tastases is predominantly one of activated effector-mem-
gineered to encode prostate specific antigen (PSA)
There was a trend to an increased CD8/CD4 T-cell ratio
ory cells, with high expression of retention integrins.
and prostate acid phosphatase (PAP) proteins and
in metastases compared to PBMC and LN (p=0.065),
Regulatory T-cells also appear prevalent, likely balancing
was evaluated in an open-label multi-center dosing
with low numbers of B cells and myeloid cells. Among
the antitumor activity of the CD8 cells. Low numbers of
+
evaluation clinical trial in non-metastatic castra-
all 14 metastases, CD8 T-cells in the MME had an
tion resistant prostate cancer (CRPC).
effector-memory phenotype: 86% CD45RA
neg
neg
+
+
+
+
CLA+ and CXCR3 T-cells likely reflect low expression
of E-selectin in intratumoral endothelium and low levels
(85-100%), and ex-
of CXCR3-cognate chemokines (CXCL9-11) produced by
range 53-99%) and 98% CCR7
BN®-HER2 and MVA-BN®-PRO who were enrolled
pressed markers of activation: 65% CD69 (17-94%),
+
neg
(7-76%), 10% CD27
+
(median,
Immune evaluation of patients treated with MVA-
neg
+
+
melanoma cells in these metastases. T-cells expanded by
in phase I clinical trials was performed to measure
28% CD28
(3-31%). FoxP3 /
vaccines, cytokines, or adoptive T-cell therapy may be
the immunogenicity of the poxvirus based immu-
CD127 cells (putative regulatory T-cells) represented
more capable of reaching their tumor targets by combi-
lo
+
notherapies. A range of assays were performed,
25 ± 6% (mean ± standard deviation) of CD4 cells
nation with interventions that upregulate endothelial E-
including phenotypic analyses by flow cytometry,
in distant metastases, but only 10 ± 4% in the TIN
selectin and intratumoral expression of chemokines for
assessment of cellular immune responses by the
(p=0.003). As expected, antigen-experienced CD8
CCR5 and CXCR3, as well as by interventions that induce
IFN-γ ELISPOT assay and a CFSE-based prolifera-
T-cells expressing CCR9 were found almost exclusively
expression of the retention integrins by T-cells that enter
161
110 | Immunomonitoring
111 | Immunomonitoring
Detection of tumour antigen specific T-cell populations in
­leukaemia: markers of good prognosis?
TransHLA – A Method for determining the HLA genotype from
RNA-Seq data
1
1
2
1
3
Suzanne Brooks , Stephanie Bonney , Evelien Smits , Cindy Lee , Dagmar Sigurdardottir ,
3
4
4
2
Hans-Georg Rammensee , Karen Pulford , Alison Banham , Viggo Van Tendeloo , Ghulam
5
1
1
1, 5,6
J. Mufti , Tim J. Elliott , Kim H. Orchard , Barbara-ann Guinn 1
Cancer Sciences Unit (MP824), University of Southampton, SO16 6YD, U.K.
2
Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute, University
of Antwerp, Belgium
3
Department of Immunology, Institute for Cell Biology, University of Tübingen, Germany
4
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Level 4 Academic
Block, John Radcliffe Hospital, Headington, Oxford, Oxon. OX3 9DU, UK
5
Department of Haematological Medicine, The Rayne Institute, London, SE5 9NU, UK
6
Division of Science, University of Bedfordshire, Park Square, Luton, LU1 3JU, UK
1, 2
1
1, 2
1
1
Sebastian Boegel , Martin Löwer , Michael Schäfer , Thomas Bukur , Jos de Graaf ,
1
1
1
1, 2
Valesca Boisguerin , Mustafa Diken , John Castle , Ugur Sahin 1
TRON – Translational Oncology at the University Medical Center Mainz, 55131 Mainz, Germany
2
University Medical Center of the Johannes Gutenberg University Mainz, III. Medical Department, Mainz, Germany
Peptide major histocompatibility complex (pMHC)
and 2 unknowns). We found that 13 acute myeloid
The human leukocyte antigen (HLA) loci are
As a proof of concept, we successfully applied the
arrays can improve our understanding of the specif-
leukaemia and one acute lymphocytic leukaemia
highly polymorphic with single nucleotide poly-
method to 50 CEU HapMap individuals yielding
ic T-cell populations involved in immune responses
patients had specific-T-cell populations recognis-
morphisms, insertions and deletions distinguish-
100% specificity and 90.8% sensitivity at a confi-
during conventional treatment and immunotherapy
ing epitopes within 8 tumour antigens including
ing 5,468 known Class I and 1,591 Class II Alleles
dence value > 0.85 and to 14 commercially avail-
clinical trials. In the longer term this could guide
PASD1, WT1126-134, MelanA and/or Tyrosinase. We
distributed in the human population (as of October
able tumor cell lines resulting in 84.5% accuracy.
clinical decisions towards more individualized,
are now investigating whether these responses cor-
2011). Besides being generally essential for the
Analysis of the remaining 15.5% of missed alleles
and potentially more effective, treatments. One
relate with response to treatment. We have devel-
immune function, the individual HLA type is im-
and mistypings reveals insights in HLA loss and
major advantage of the technique is its ability to
oped a robust method for the simultaneous analysis
portant e.g. for allotransplantation of organs and
downregulation in tumor cells.
simultaneously analyse multiple T-cell populations
of T-cell populations in leukaemia patients, which
prevention of transplant rejection.
+
using small numbers of “untouched” CD8 T-cells
can indicate a short-list of T-cell populations for
Next generation sequencing (NGS) is a rapidly
(approximately 0.8 - 1.2 x 10 CD8 T-cells/array). further investigation of T-cell function minimising
evolving technique for parallel sequencing of bil-
In addition pMHC arrays use very small amounts of
reagent and sample use.
lions of nucleotides. With the associated per-nu-
6
+
th
pMHC per spot (1ng, 1/1,000 of that used in flow
cleotide costs constantly decreasing, NGS becomes
cytometry) and can simultaneously analyse a large
available for routine clinical applications. Espe-
number of T-cell populations without haplotype
cially re-sequencing of a panel of targeted genomic
+
restriction. CD8 T-cells were negatively isolated
regions or transcripts for individual patients
from leukaemia patients, lipophillically dyed with
becomes cheap and time efficient.
DiD and incubated with pMHC arrays printed with
Here we present a method for obtaining an individ-
more than 50 tumour-associated antigen (includ-
ual HLA type using NGS transcriptome (RNA-Seq)
ing WT1 and Proteinase 3) and viral epitopes (in-
data. This method consists of a mapping and a re-
cluding CMV and Flu). Positive scoring of T-cells
finement step. In the mapping step, RNA-seq reads
bound to pMHC were only made when T-cells were
are aligned against a reference database contain-
consistently bound to the same pMHC spots in two
ing the coding sequences of all known HLA alleles.
distinct regions on the array. We have analysed
In the refinement step, the loci are classified into
41 samples from 33 patients (23 acute myeloid
homo- and heterozygosity, followed by the compu-
leukaemia including eight follow-up, 3 acute lym-
tation of a confidence score of all predicted alleles.
phocytic leukaemia, 5 chronic myeloid leukaemia
162
163
112 | Immunomonitoring
Predictive biomarkers for treatment success of the therapeutic
renal cell cancer vaccine IMA901
1
1
2
2
1
Andrea Mahr , Jens Fritsche , Alexander Zien , Stephan Brock , Vlatka Stos-Zweifel ,
1
1
1
1
1
Helen Hörzer , Phillip Müller , Regina Mendrzyk , Norbert Hilf , Steffen Walter ,
1
1
Harpreet Singh-Jasuja , Toni Weinschenk
1
Immatics biotechnologies GmbH, Tübingen, Germany
2
Molecular Health GmbH, Heidelberg, Germany
Therapeutic anti-cancer vaccines show the promise
with an “elastic net” regularizer. Generalizability
of combining meaningful clinical efficacy with low
was evaluated by a leave-one-out cross-validation
toxicity due to their cancer-specific mechanism of
approach. To distinguish between predictive and
action. However, the induction of immune respons-
purely prognostic markers, a control arm is re-
es and t heir translation into enhanced survival can
quired. As the study arm receiving CY (+CY arm)
be hampered by the compromised immune system
performed better with respect to clinical efficacy
observed in cancer, such that a fraction of patients
and translation of T-cell responses into improved
do not show benefit from vaccination. This study
survival, markers were selected based on their se-
aimed at identifying pre-treatment biomarkers pre-
lective association with overall survival in the +CY
dictive for the success of treatment with the multi-
but not in the –CY arm.
peptide based vaccine IMA901, which was designed
The analysis yielded a set of 20 variables which was
to induce specific T-cell responses against antigens
predictive for overall survival after the combined
found on renal cell carcinoma (RCC).
treatment with IMA901 and CY. The predictive
A phase II clinical study (study code IMA901-202)
performance of the marker sets identified in the
was conducted in 68 (64) intent-to-treat (per-pro-
phase II study will be tested in an ongoing phase
tocol) metastatic RCC patients. Patients were ran-
III clinical study.
New Targets & New Leads
domized to two arms, receiving or not receiving a
single dose of cyclophosphamide (CY) before vaccination.
Univariate statistical analysis led to the identification of two biomarkers, apolipoprotein A-I and
CCL17/TARC, which were combined in a score
model predictive for overall survival and immune
response to the vaccine (as published at the last
CIMT Meeting). Here, results of the multivariate
analysis are shown. More than 200 pre-treatment
variables were analyzed for a possible association
with overall survival by multivariate Cox regression. As there were many more variables than patients, careful modeling was required to avoid overfitting. Therefore, the Cox model was combined
164
165
113 | New Targets & New Leads
114 | New Targets & New Leads
Regression of metastatic melanoma by targeting melanoma
stem cells
EpCAM-targeting antibodies for the treatment of a murine
model of spontaneous gastric cancer
1
2
1
3
1
1
1
2
Patrick Schmidt , Max Schlaak, Andreas A. Hombach , Christopher Bangard,
2
2
2, 4
1, 4
Peter Kurschat, Paola Zigrino , Cornelia Mauch & Hinrich Abken
Jonas Henkel , Julius Steffen , Simon Graßmann , Wolfgang Zimmermann ,
1
1
1
Carole Bourquin , Stefan Endres and Sebastian Kobold 1
Tumorgenetics, Department I of Internal Medicine
1
2
Department of Dermatology and Venerology
Division of Clinical Pharmacology, Center of Integrated Protein Science Munich (CIPS-M),
­Department of Medicine IV, Ludwig – Maximilians Universität, Munich, Germany
3
Institute for Radiological Diagnostics, University Hospital Cologne and CIO Cologne-Bonn
2
Tumor Immunology Laboratory, LIFE Center, Ludwig-Maximilians-Universität, Munich, Germany
4
Center for Molecular Medicine Cologne, University of Cologne, Germany
Current paradigms in cancer therapy attempt to
Background: The epithelial cell adhesion molecule
Remarkably, survival in this therapy-resistant
eliminate all malignanT-cells of a tumor lesion, the
(EpCAM) is highly expressed in gastrointestinal
model was prolonged by 11 days (p= 0.06).
cancer stem cell (CSC) paradigm, however, predicts
malignancies. There, EpCAM is suggested to be a
Conclusions: EpCAM-targeting antibodies dem-
that tumors are maintained by a few, so far less
marker of cancer stem cells and to be involved in
onstrate good cytotoxicity in an in vitro model of
identified cancer stem cells. Here we demonstrate
processes such as metastasis. These characteris-
gastric cancer. First in vivo data indicate that our
that specific elimination of a less than 2% subset
tics render EpCAM a highly interesting target for
antibody specifically targets cancer cells in a spon-
of melanoma cells eradicates transplanted human
antibody-based immunotherapies, since EpCAM is
taneous model of gastric cancer which is known to
melanoma biopsies in a mouse model without tar-
additionally present on the cell surface. To this end
be therapy-resistant. This data is of high relevance,
geting the bulk of tumor cells. The melanoma cell
new antibodies need to be identified and tested in
as in vitro cytotoxicity as well as penetrance and
subset is selectively and specifically eliminated
appropriate preclinical methods.
accumulation into a spontaneous tumor are man-
from established tumor lesions by adoptive transfer
Methods: We investigated two EpCAM-specific an-
datory for further in vivo use. Our data suggest that
of cytotoxic T-cells redirected by a chimeric antigen
tibodies in a spontaneous model of gastric cancer,
EpCAM represents an attractive target for the treat-
receptor. Targeted elimination of the minority of
the
mouse
ment of gastric cancer and that specific antibodies
CD20 melanoma cells eradicated melanoma lesions
(SV40) both in vitro and in vivo. Antibodies were
may overcome local and systemic tumor resistance.
in the long-term despite the non-targeted tumor cell
characterized by Biacore-measurement. Expres-
mass. Targeting of any random cancer cell subset
sion of EpCAM was addressed by flow cytometry
was not effective. Based on these pre-clinical data
and immunofluorescence.
we attempted to eliminate those cells in a patient
Results: Antibodies with murine or rat backbone
+
CEA424-SV40-T
antigen-transgenic
with progressing, chemotherapy-refractory meta-
had affinities for EpCAM in the nanomolar range
static melanoma by lesional injections of the anti-
and recognized recombinant and naturally pro-
CD20 therapeutic antibody rituximab. Although
cessed EpCAM protein. Both the primary tumor
the frequencies of CD20 melanoma cells within
+
and a cell line established from the SV40-mouse
the tumor lesions were initially about 2% and the
expressed EpCAM at high levels (range 60 – 100%
bulk of tumor cells did not express CD20, rituximab
of all tumor cells). In vitro, both antibodies had
treatment produced lasting remission accompanied
cytotoxic activity through the classical way of com-
by a decline of the melanoma serum marker S-100
plement (range 20 – 40% of cell lysis) and through
to physiological levels. Apart from B cell elimina-
antibody-mediated
tion and decline in gammaglobulin levels, no grade
(range 50 – 80% of cell lysis). When applied in vivo
3/4 toxicity related to treatment was observed. Data
EpCAM-specific antibody was found evenly dis-
+
cell-dependent
cytotoxicity
provide the first clinical evidence of CD20 "mela-
tributed in the EpCAM-positive tumor as well as
noma sustaining cells" and highlight the potency
in EpCAM-expressing tissues. Distribution of the
of selective cancer cell targeting in the treatment
antibody tightly correlated with the expression of
of melanoma.
EpCAM in the given tissue. Next, antibodies were
applied in a therapeutic setting in the SV40-mice.
166
167
115 | New Targets & New Leads
116 | New Targets & New Leads
Development of a novel modular cell targeting system for
­immunotherapy of acute myeloid leukemia
HLA-restricted CD4+ T-cell epitopes derived from cancer-retina
antigen PDE6 alpha as potential tools for T-cell based immunotherapy approaches
1
1
1
2
1
1
1
1
2
1
Claudia Arndt , Anja Feldmann , Marc Cartellieri , Malte von Bonin , Stefanie Koristka ,
1
1
2
2
1
Irene Michalk , Slava Stamova , Martin Bornhäuser , Gerhard Ehninger , Michael Bachmann Maria Jęsiak , Wolfram Osen , Adriane Gardyan , Sabine Soltek , Elke Dickes ,
2
1
Alexandr V. Bazhin , Stefan B. Eichmüller
1
Medical Faculty ‘Carl Gustav Carus’, Institute of Immunology, Fetscherstraße 74, 01307 Dresden,
Germany
1
Division of Translational Immunology, DKFZ German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
2
University Hospital ‘Carl Gustav Carus’, Medical Clinic and Polyclinic I, Fetscherstraße 74,
01307 Dresden, Germany
2
Department of General Surgery, University Hospital Heidelberg, 69120 Heidelberg, Germany
168
+
Conventional tumor therapies often do not achieve
systems. In vitro studies using CD33
the complete eradication of metastatic cancer cells.
lines as well as patient-derived AML blasts clearly
AML cell
Thus, novel approaches are required to enhance
demonstrate that T-cells can be specifically activat-
the effects of standard treatment modalities. One
ed for highly potent tumor cell lysis by both systems
Melanoma originates from melanocytes and is a
CD4
compelling immunotherapeutic strategy is based
already in picomolar concentrations. Moreover,
fast progressing tumor which is known to be highly
Therefore, HLA-transgenic (tg) mice expressing
on recombinant bispecific antibodies (bsAb). BsAb
our data confirm that not only CD8 but also CD4
resistant to many therapies. Although it has been
HLA-DRB1*0301 (DR3tg) were immunized with
consist of the variable light and heavy chains of
T-cells can be specifically recruited for the elimi-
proven to be very immunogenic, the immune re-
an expression plasmid (pcDNA3.1-PDE6a) encod-
two mAb recognizing different antigens. Due to
nation of malignanT-cells in a target-specific and
sponse of the patients is often not able to fight back
ing human (xenogenic) PDE6a, followed by ex vivo
the simultaneous targeting of a tumor associated
TCR-independent manner, even though the onset of
and eliminate all tumor cells.
analyses of the PDE6a-specific T-cell responses
with a synthetic peptide library covering the
+
+
+
T-cell responses in melanoma patients.
antigen (TAA) and the activating CD3-complex,
killing via CD4 T-cells is delayed. The antitumor
A number of various melanoma associated anti-
bsAb are able to recruit effector T-cells specifically
response of both Ab-based systems was further
gens are well known. It has been recently shown
entire PDE6a protein. Applying this strategy, two
HLA-DRB1*0301 restricted epitopes were identi-
+
into tumor tissues. The bsAb-mediated cross-link-
evaluated in vivo. Co-injection of CD33
tumor
that a new class of tumor antigens, called cancer-
age triggers a polyclonal T-cell activation that leads
cells with T-cells in the presence of the bsAb or
retina antigens (CRA), apart from being expressed
fied and CD4
to an effective killing of the recognized targeT-
the modular system in immunodeficient NSG mice
in healthy retina (and participating there in the
sponding epitopes were established. These murine
cells. First clinical trials have proven the efficiency
resulted in a significantly lower bone marrow chi-
visual transduction pathway), can be expressed
CD4 T-cell lines will be tested for recognition of
of this approach for tumor treatment.
merism compared to non-treated control animals.
at the mRNA and protein levels in melanoma.
human HLA-matched targeT-cells: firstly, when
For redirection of T-cells to acute myeloid leuke-
Taken together, our in vitro and in vivo data under-
This aberrant expression of CRA is capable to
loaded with antigenic peptide; and secondly, when
mia (AML) blasts we established a novel human-
line the high potential of both the direct cross-link-
elicit humoral and cellular immune responses in
expressing the target antigen endogenously. This
ized bsAb targeting CD33 as a promising leukemia-
ing bsAb CD33-CD3 and the novel modular system
melanoma patients. Among these CRA, cGMP-
strategy would prove that the epitopes identified in
associated antigen. However, one major obstacle of
as powerful tools for an antigen-specific immuno-
phosphodiesterase 6 alpha (PDE6a) was found to
HLAtg mice actually represented natural process-
bsAb is their complex and time-consuming develop-
therapy of AML patients. In particular, the modular
be expressed in many melanoma cell lines as well
ing products also in the human system.
ment. Therefore, we additionally developed a flex-
system provides a series of advantages compared
as in tumor tissues of both, the human and murine
Furthermore, the analysis of peripheral blood
ible modular cell targeting system composed of two
to classical bsAb. Applying the modular system to
system. Therefore PDE6a could be considered as
mononuclear cells from HLA-DR3 tumor patients
different Ab components. The first component is a
other malignancies only requires the exchange of
a suitable target antigen for T-cell based immuno-
for the presence of CD4
single-chain fragment variable (scFv)-based target-
the targeting module while the effector module can
therapy approaches against melanoma, and possi-
same epitopes identified in HLA-transgenic mice is
ing module that comprises a binding arm for the
be maintained. The construction of new scFvs as
bly against further tumor entities such as pancre-
planned. These T-cells will be expanded and tested
TAA CD33 and a short peptide epitope. The second
novel targeting modules is far less time-consuming
atic carcinoma where PDE6a expression was also
for their reactivity against HLA-matched human
component (effector module) is a bsAb with antigen
than the development of new bsAb, which always
detectable.
melanoma cell lines in vitro.
binding specificities for the CD3-complex on T-cells
requires a series of individual optimization steps.
The essential role of tumor antigen-specific CD4
and for the peptide epitope of the targeting module.
Furthermore, by using a targeting module recog-
T-cell responses for efficient tumor eradication has
Together both molecules are able to form protein
nizing two different antigens the modular system
been demonstrated in various tumor models and
complexes that act in a similar way as classical bsAb.
offers the possibility of a multi-specific targeting
in clinical settings. Thus, our aim was to identi-
In the present work we compare the functional-
which may improve target specificity and therefore
fy novel CD4 T-cell epitopes that could be used
ity and efficiency of both Ab-based cell targeting
increase the success of an immunotherapy.
for the induction and detection of tumor-reactive
+
+
T-cell lines specific for the corre-
+
+
+
T-cells specific for the
+
+
169
117 | New Targets & New Leads
118 | New Targets & New Leads
Targeting mutated and overexpressed tumor antigens in cancer
immunotherapy
Mutated BRAF and NRAS proteins as possible targets for the
immunotherapy of melanoma
1
1
1
1
Sandra Höfflin , Beatrice Schuler-Thurner , Stefanie Gross , Gerold Schuler , Niels
1,*
1 *
Schaft , and Jan Dörrie ,
Sabrina Prommersberger, Beatrice Schuler-Thurner, Stefanie Gross, Gerold Schuler,
*
*
Niels Schaft , and Jan Dörrie
*
J.D. and N.S. share senior authorship
Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany
1
Department of Dermatology, Universitätsklinikum Erlangen, Erlangen, Germany
*
Identifying new candidate antigens as potential targets
mutated peptide pool (9 aa long), produced only low
for cancer immunotherapy opens new chances for
quantities of cytokines. Elispot analyses revealed an-
tumor patients. Mutations in GNAQ and GNA11, which
tigen-specific IFNγ-production by T-cells stimulated
commonly occur in particular codons and emerge in a
with PBMCs, which were transfected with the wild
majority of uveal melanomas, could create such new
type or mutated forms of the antigen, while co-cultiva-
antigens, which emerge in a majority of uveal melano-
tion with PBMCs loaded with a GNAQ-peptide-pool of
The valine to glutamic acid mutation of BRAF at
antigen-specific IFNγ-production by intracellular
mas.. Other self-proteins, like the Wilms’ tumor (WT1)
9-mers could not induce specific cytokine production.
codon 600 and the mutation of NRAS at codon 61
cytokine staining and Elispot assays. In these anal-
protein, are overexpressed in many kinds of adult leu-
Considering MFTC analyses of the GNA11 stimulations,
both lead to a constitutive activation of the RAS-
yses we stimulated the T-cells with electroporated
kemia and solid tumors.
an increase in the fraction of CD8 T-cells producing
RAF-MEK-ERK-MAP kinase pathway. Both muta-
DC, peripheral blood mononuclear cells (PBMC)
We analyzed the mutation frequency of GNAQ and
TNF and INFg was detected with antigen-specifically
tions are frequently found in melanoma cells, since
or non-adherent fraction cells (NAF). While most
GNA11 (Q209P or Q209L) in melanoma cultures cells
stimulated cells. As with GNAQ, no clear difference in
they lead to the transduction of survival and pro-
of the stimulations did not lead to any notewor-
from patients and melanoma cell lines via PCR and
the immunogenicity of wild type or mutated antigens
liferation signals.
thy IFNγ release, the T-cells of two donors showed
subsequent sequencing. These mutations exclusively
could be seen. CD8 T-cells, stimulated with mDCs that
Based on the high frequency of these oncogenic
high IFNγ releases after stimulation with BRAF or
occurred in cells of uveal melanoma patients (1/2, and
had been loaded with a GNA11-peptide pool of 9-mers,
mutations in malignant melanoma (about 50% for
BRAFV600E-presenting cells.
1/2, respectively), but could not be detected in other
produced merely poor quantities of cytokines during
BRAF and 20% for NRAS) and their exclusive oc-
We also performed a similar experiment by using
melanoma cell lines (0/21).
stimulations. Elispot analyses of GNA11 stimulations
currence in tumor cells, the mutated BRAF and
NRAS, NRASQ61K and NRASQ61R RNA for elec-
To investigate whether the mutated versions of the
revealed inconclusive data.
NRAS proteins may be a good target in the immu-
troporation. After the second stimulation antigen-
antigens GNAQ and GNA11 are more immunogenic
Regarding WT1, the immunogenicity of WT1 was com-
notherapy of this cancer. The aim of a therapy ad-
specific IFNγ production was detected in some ex-
+
dressing these mutations would be the activation
periments by intracellular staining.
+
+
than their wild type forms, CD8 T-cells were stimu-
+
pared to a WT1-DCLamp construct. Therefore, CD8
+
lated three times with autologous mature dendritic cells
T-cells were stimulated three times with autologous
of CD8 T-cells in the patient which are specific for
Furthermore, we developed a simple and rapid
(mDCs), which had been equipped with wild type or
mDCs, which had been transfected with WT1 or WT1-
the mutated forms of BRAF and / or NRAS.
method to examine melanoma cell lines for the spe-
mutated antigens by RNA-electroporation. Success-
DCLamp. Successful transfection was demonstrated by
Here we want to test whether there are CD8 T-cells
cific BRAF and NRAS mutations. For that purpose,
ful transfection of the mDCs was verified by intracel-
intracellular FACS analyses. The antigen-specific activ-
in the blood of healthy donors, which are able to
we isolated RNA out of the cells, performed cDNA-
lular FACS analyses. The antigen-specific activity of
ity of the stimulated CD8 T-cells was then determined
recognize and react specifically to BRAFV600E,
synthesis and -amplification. This DNA was se-
+
+
+
the stimulated CD8 T-cells was determined by Multi
by MFTC analyses, with antigen-loaded mDCs used as
NRASQ61K or NRASQ61R-presenting dendritic
quenced with particular primers attaching on both
Functional T-cell Assay (MFTC), where antigen-loaded
targeT-cells in short-time stimulations. Furthermore,
cells (DC). We also want to investigate if the
sides of the mutation site. Up to now, we found
mDCs were used as targeT-cells for short-time stimula-
IFNγ-Elispot was used to test the functionality of the
mutated versions of these antigens are more im-
that 10 out of 18 cell lines carry the BRAF
stimulated CD8 T-cells, using antigen-loaded PBMCs
munogenic than their wild-type versions.
tation while 4 out of 15 cell lines are mutated in
CD8 T-cells was tested by IFNγ-Elispot with antigen-
as targets. MFTC analyses of the WT1 stimulations re-
Therefore, we equipped mature DC with the wild
NRAS at codon 61 (2x NRASQ61R, 1x NRASQ61L,
loaded peripheral blood mononuclear cells (PBMCs)
vealed an increase in the percentage of CD8 T-cells that
type or the mutated BRAF antigen by RNA-elec-
1x NRASQ61K). In none of these cell lines we found
used as targets.
produced TNF and IFNγ, when the T-cells were stimu-
troporation. Additionally, we transfected the DC
both mutations at the same time - the mutation in
lated by antigen-transfected mDCs. However, the first
with constitutively active forms of IKKa and IKKb
one of the genes usually excludes a mutation in the
results do not assign a distinct form of the protein more
for enhanced stimulatory capacity. Afterwards, au-
other one.
tions. Additionally, the functionality of the stimulated
+
MFTC analyses of the GNAQ stimulations revealed an
+
increase in the percentage of CD8 T-cells that pro-
+
+
+
V600E
mu-
duced TNF and IFNγ, when stimulated with antigen-
immunogenic than the other. Corresponding results
tologous CD8 T-cells were stimulated for one week
The next steps will be to test further melanoma cell
transfected mDCs. However, no statement could be
were obtained by Elispot analyses.
with the electroporated DC, and then re-stimulated
cultures for mutations in NRAS in BRAF. In our
made concerning the immunogenicity of the wild type
Further stimulations will be performed with the anti-
a second and a third time with the same stimula-
stimulation experiments we have started to opti-
gens under improved conditions and further cell lines
tor cells. To analyze the expansion and function of
mize the experimental and the readout conditions
+
compared to mutated versions of GNAQ. CD8 T-cells
stimulated with mDCs, which were loaded with a
170
J.D. and N.S. share senior authorship
will be analyzed for mutations in GNAQ and GNA11.
+
antigen-specific CD8 T-cells, we measured their
to get more distinct results.
171
119 | New Targets & New Leads
120 | New Targets & New Leads
Natural HLA ligands provide novel T-cell epitopes
for ­immunotherapy of ovarian carcinoma
Identification of new tumor specific HLA-Ligands –
Separating tumor and stroma origin
1
2
1
3
1
1
1
2
3
3
Janet Peper , Helen Hörzer , Stefan Stevanovi , Richard Schäfer , Hans-Georg Rammensee 2
and Brigitte Gückel Heiko Schuster , Janet Peper , Philipp Wagner Jörg Hennenlotter Arnulf Stenzl 1
and Stefan Stevanović 1
Department of Immunology, Institute for Cell Biology, University of Tübingen, Germany
1
2
Department of Obstetrics and Gynecology, University Hospital Tübingen, Germany
Department of Immunology, Institute for Cell Biology, University of Tübingen,
Auf der Morgenstelle 15, 72076 Tübingen
3
Institute of Clinical and Exp. Transfusion Medicine, University Hospital Tübingen, Germany
2
Department of Obstetrics and Gynecology, University hospital Tübingen, Calwerstraße 7,
72076 Tübingen
3
Department of Urology, University hospital Tübingen, Hoppe-Seyler-Str 3, 72076 Tübingen
172
Late diagnosis and resistance to chemotherapy are
overexpressed in OvCa and associated with poor
Renal cell carcinoma and ovarian cancer are both
to verify that this can also be seen directly on the
the main reasons for the high mortality among
prognosis. Several substances targeting HDACs
characterized by poor prognosis and high mortality
level of the HLA derived peptides we started again
women suffering from ovarian carcinoma (OvCa).
are already in clinical trials. In vitro priming of
due to late diagnosis and high resistance to conven-
from a bulk tissue derived single cell suspension
New strategies to circumvent this drug resistance
healthy PBMCs induced T-cell responses against
tional chemo- and radiotherapy. Curative treatment
but this time used MACS-technology to separate
are urgently needed. One approach is to develop
the HDAC1 peptide in eleven of twenty-one donors,
for both cancers is often only possible through sur-
tumor from stroma cells. As expected tumor cells
immunotherapies, including peptide-based cancer
as determined by positive intracellular stainings
gical resection at an early non-metastatic stage.
and surrounding stroma cells showed a completely
vaccines which should induce tumor-specific
for IFNγ, TNFα, IL-2, CD107a and MIP-1β. In ad-
Looking for new and effective therapeutic options
distinct pattern of HLA presented peptides. With
T-cells. Several OvCa-associated antigens have
dition, HDAC1-primed T-cells lysed peptide-loaded
a lot of effort has been put into the development of
this approach we have identified new tumor spe-
+
already been identified by expression profiling.
as well as unloaded HLA-A*02
tumour cells in
a specific immunotherapy, aiming at the in vivo in-
cific MHC ligands derived from already established
Further knowledge regarding MHC-presented pep-
chromium release assays while HLA-A*02-negative
duction of a tumor-directed immune response. Our
cancer related antiges (e.g. TMPRSS3) and could
tides derived from tumour-associated antigens is
cells were not lysed. Additionally, this natural HLA
group is especially interested in the identification of
also reevaluate previously found peptides that were
needed for triggering specific T-cell responses.
ligand has been identified on three further OvCa,
tumor specific MHC ligands that can be used for the
formerly claimed to be tumor specific and are now
The purpose of this study is to develop a multiva-
four renal cell carcinomas, one breast cancer and
development of a multivalent peptide vaccine tar-
more likely to originate from the tumor stroma.
lent peptide vaccine targeting OvCas.
one prostate cancer sample. Moreover this peptide
geting different tumor types. For several years we
Up to now we have analysed MHC class I ligands of
is also a natural HLA-A*02 ligand of HDAC2, allow-
and other groups have relied on bulk tumor tissue
seven OvCa samples. This was performed by liquid
ing the simultaneously targeting of two different
to perform HLA ligand extraction and analysis by
chromatography coupled mass-spectrometry. Pep-
HDACs associated with tumorigenesis.
mass spectrometry. However information about the
tides from established tumour-associated antigens
According to our experimental data the novel T-cell
amount of isolated MHC molecules directly derived
(TAA) and peptides from proteins which might be
epitope of HDAC1 and HDAC2 will be included in
from tumor cells has not yet been obtained.
involved in tumourigenesis or angiogenesis were
peptide-based vaccination trials.
In order to get a comprehensive view of the distri-
selected for in vitro priming performed with PBMCs
bution of MHC expression within tumor tissue we
(peripheral blood mononuclear cells) from healthy
used multi color flow cytometry to analyze the cel-
blood donors. T-cells were isolated by magnetic
lular composition and quantified the MHC expres-
activated cell sorting and stimulated with peptide-
sion of specific cell subsets. Single cell suspensions
pulsed autologous dendritic cells and B cells.
of fresh tissue samples were prepared by enzymatic
Altogether we identified 1032 HLA ligands from
digestion and stained with respective antibodies to
seven different OvCa samples, whereas 22 of these
distinguish between leukocytes, tumor cells, en-
peptides were derived from TAAs. Up to know, in
dothelial cells and fibroblasts. Our results show that
vitro priming of these candidate peptides revealed
MHC molecules expressed by tumor cells represent
that eight peptides are immunogenic.
only a small part of the overall MHC content. These
The most promising epitope we found is an HLA-
results emphasize the importance of using specific
A*02:01 restricted peptide derived from HDAC1
cell subsets instead of bulk tissue for the identifica-
(histone deacetylase 1), an established TAA highly
tion of new tumor specific HLA ligands. In order
173
121 | New Targets & New Leads
122 | New Targets & New Leads
HPV16 E6 and E7 T-cell epitope identification by mass
­spectrometry
Identification of NPM-ALK-reactive T-cells in children with
NPM-ALK-positive anaplastic large cell lymphoma (ALCL)
1
1
2
1
1
1
2
1
2
Renata Blatnik , Jan Winter , Uwe Warnken , Stephanie Hoppe , Agnieszka K. Grabowska ,
3
1
3
2
1
Sebastian Link , Martin Wühl , Thomas Ruppert , Martina Schnölzer , Angelika B. Riemer Sebastian Werner , Vijay Singh , Christine Damm-Welk , Volker Lennerz ,
1
1
2
Wilhelm Wössmann , Alfred Reiter , Thomas Wölfel 1
Immunotherapy and -prevention, German Cancer Research Center (DKFZ), Heidelberg, Germany
1
2
Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
Department of Pediatric Hematology and Oncology, Justus-Liebig-University, Giessen, Germany
3
Core Facility for Mass Spectrometry and Proteomics, ZMBH, Heidelberg, Germany
2
III. Med. Klinik, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
To rationally design therapeutic cancer vaccines,
174
the presence of candidate epitopes was analyzed
2
3
+
Background: ALK anaplastic large cell lymphoma
assay on day 19. To assess both the antigen reactiv-
it is important to know which T-cell epitopes are
by nano-UPLC-ESI-MS and -MS mass spectrom-
(ALCL) represents an ideal model to characterize the
ity and the HLA restriction, responder T-cells were
present on cancer cells. Malignant transformation
etry. To this end, resulting spectra were compared
mechanism of immune responses against tumor-
tested for the recognition of COS-7 cells co-trans-
affects the cellular antigen processing machinery,
to reference spectra of synthetically produced pep-
specific mutated antigens. ALCL is the third most
fected with the respective donor´s individual HLA
thus not every epitope derived from intracellular
tides of interest.
common Non-Hodgkin´s lymphoma in children and
class I alleles and NPM-ALK cDNA. pp65 IVT-RNA
proteins is necessarily presented by MHC mol-
Binding of the majority of predicted peptides to the
adolescents (1) and expresses tumor-specific onco-
stimulations were performed in parallel as a posi-
ecules on the cancer cell surface.
respective HLA allele was verified in the cellular
genic ALK-fusion proteins resulting from chromo-
tive control in seropositive blood donors.
Up to now, T-cell epitopes have mostly been defined
assays. Several novel HPV16 MHC binders were
somal translocations. The most common transloca-
Results: In two out of three patients with NPM-ALK
+
+
by indirect methods, such as screening peptide li-
identified. Mass spectrometry analysis was estab-
tion t(2;5) fuses the anaplastic lymphoma kinase
ALCL NPM-ALK-reactive CD8 T-cells could be en-
braries for MHC binders, and subsequent lympho-
lished and validated with HLA-A2. To assess the
gene (ALK) to the nucleophosmin gene (NPM) (2).
riched and detected by mRNA stimulation in blood
+
cyte reactivity and cytotoxicity assays. However,
minimal cell number required for MS analysis, a
Patients with ALK ALCL can mount an antibody
samples collected one to eight years after diagno-
all these assays with externally added peptides
series of immunoprecipitation (IP) samples were
response against ALK. Anti-ALK antibody titers at
sis. In both patients, recognition of NPM-ALK was
+
do not answer the question whether a candidate
prepared from the HLA-A2
HPV16-transformed
diagnosis inversely correlate with clinical stage, cir-
restricted by HLA-C alleles. No NPM-ALK-reactive
epitope is naturally processed and presented on
cell line CaSki. For reliable detection of a low-abun-
culating tumor cells and the cumulative incidence
T-cells could be detected in three healthy controls.
the malignanT-cell. This can now be elucidated
6
dance HPV epitope, 5x10 CaSki cells are required.
of relapses (3), suggesting a role of the immune re-
Conclusion: NPM-ALK-reactive CD8 T-cells were
by using highly sensitive mass spectrometry ap-
To perform HLA-A2 IPs of other HPV16-trans-
sponse in tumor control. Using reverse immunology
successfully expanded and detected in ALCL pa-
+
+
+
proaches.
formed cell lines, the expression level of HLA-A2
approaches, CD8 as well as CD4 T-cell responses
tients via mRNA stimulation. Using autologous
For this study, we chose high-risk HPV-driven
was determined in eleven HPV16-transformed cell
against NPM-ALK-derived peptides, as predicted by
FastDCs transfected with full-length IVT-mRNA as
cancers as a model system, such as cervical or
lines by FACS staining. InpuT-cell numbers for IPs
binding score algorithms, were detected in both pa-
antigen-presenting cells allows to comprehensively
certain oropharyngeal carcinomas. The induction
were adjusted relative to CaSki HLA-A2 expression
tients and healthy individuals (4,5,6). These analy-
exploit T-cell reactivity against NPM-ALK, because
and maintenance of the malignant phenotype of
levels. In all HLA-A2 HPV16 cell lines tested, the
ses were limited to few HLA alleles and thereby did
all potential peptide epitopes are included in the
these tumors are dependent on two viral oncopro-
HPV epitope E711-19 was found to be naturally pro-
not cover the full immunogenicity of NPM-ALK.
context of the complete individual HLA repertoire.
teins, E6 and E7, which are therefore present in all
cessed and presented on the cell surface, whereas
Moreover, they did not proof endogenous process-
In addition, this strategy guarantees that the identi-
stages of HPV-driven cancers. We here aimed to
other epitopes were not constantly present.
ing of the respective peptides. To overcome these
fied target epitopes are indeed processed and trans-
assess the presence of HPV T-cell epitopes restrict-
In conclusion, we show that ascertaining the actual
limitations, we decided to use autologous dendritic
ported to the cell surface.
ed by four major MHC class I supertypes, namely
cellular presentation of HPV T-cell epitopes is fea-
cells transfected with in vitro-transcribed (IVT),
Perspectives: NPM-ALK is a promising candidate
HLA-A1, A2, B7, and B15, on HPV16-transformed
sible. The finding that not every possible epitope
full-length mRNA encoding NPM-ALK to identify
target antigen for active and passive immune inter-
cells.
is necessarily presented highlights the importance
NPM-ALK-specific T-cell responses.
ventions complementing and consolidating standard
Prospective epitopes from the HPV16 E6 and E7
of determining the presence of an epitope on the
Methods: CD8 T-cells of patients with ALK ALCL
chemotherapy of ALK ALCL. Findings and experi-
proteins for the above MHC supertypes were iden-
targeT-cell before initiating vaccine design. Verified
and of healthy donors were stimulated under qua-
ences with NPM-ALK can perhaps be extrapolated to
tified by high-throughput in silico epitope predic-
epitopes are also important for immunomonitoring
si-limiting dilution conditions with autologous
other cancers driven by ALK-fusion proteins.
tions, employing several web-based prediction
purposes.
dendritic cells [FastDCs, (7)]. Prior to stimulation,
+
+
+
+
algorithms. These peptides were tested for MHC
FastDCs were transfected using the Amaxa Nu-
binding in competition-based cellular binding
cleofection system with IVT-mRNA encoding full-
assays on B-LCL of the respective HLA type. Sub-
length NPM-ALK. After two restimulations on day
sequently, MHC-peptide complexes were immuno-
7 and 14 under similar culture conditions, T-cell
precipitated from HPV16-transformed cells, and
responses were analyzed with an IFN-γ ELISPOT
+
(1) Burkhardt et al. Br J Haematol 2005 Oct;131(1):39-49;
(2) Brugières et al. J Clin Oncol. 2009 Feb 20;27(6):897-903;
(3) Ait-Tahar et al. Blood. 2010 Apr 22;115(16):3314-9; (4)
Passoni et al. Blood. 2002 Mar 15;99(6):2100-6; (5) Ait-Tahar
et al. Int J Cancer. 2006 Feb 1;118(3):688-95; (6) Ait-Tahar et
al. Cancer Res. 2007 Mar 1;67(5):1898-901; (7) Dauer et al. J
Immunol Methods.2005 Jul; 302 (1-2): 145-55.
175
123 | New Targets & New Leads
124 | New Targets & New Leads
Attenuation of Lethal Semliki Forest Virus Neurovirulence in
Mice by Neuronal microRNA Targeting
Siglec-7 and -9 on Natural killer cells and their ligands on tumor cells are novel inhibitory regulators of human NK cell
functions in vitro and of tumor cell killing in vivo
1
2
1
Miika Martikainen , Erkko Ylösmäki , Ari Hinkkanen , Kalle Saksela
2
1
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland
2
Haartman Institute, University of Helsinki, Helsinki, Finland
MicroRNAs (miRNAs) are small non-coding RNA
applications by specific miRNA targeting with po-
molecules that have important regulatory roles in
tential of concomitant detargeting from critical pe-
gene expression by targeting mRNAs for cleavage
ripheral tissues.
or translational repression. The miRNA machinery
has recently been successfully exploited to modify
tropism of both RNA and DNA viruses to prevent
viral replication in specific tissues.
Acknowledgements: The study was supported by the
Academy of Finland, the Cancer Center of Eastern Finland,
and Kuopio University Hospital
1
2
1
1
1
Jandus Camilla , Chijioke Obinna , Wehrli Marc , Frias Boligan Kayluz , Liu He , Conus
1
3
1
4
Sébastien , Demoulins Thomas , Zangenmeister-Wittke Uwe , Stroka Deborah ,
5
1
6
2
1
Hunger Robert , Simon Hans-Uwe , Romero Pedro , Münz Christian , von Gunten Stephan 1
Institute of Pharmacology, University of Bern, Bern, Switzerland
2
Department of Viral Immunobiology, Institute of Experimental Immunology, University of
Zurich, Zurich, Switzerland
3
Institute of Virology and Immunoprophylaxis (IVI), Mittelhäusern, Switzerland
4
Visceral and Transplantion Surgery, Department of Clinical Research, University of Bern, Bern,
Switzerland
5
Departement of Dermatology, University Hospital of Bern, Bern, Switzerland
6
Division of Clinical Onco-Immunology, Ludwig Center of Cancer Research of the University of
Lausanne, Lausanne, Switzerland
Replicative VA7 vector based on strain A7(74) of
Semliki Forest virus (SFV) has emerged as a promising tool for oncolytic virotherapy particularly
176
for brain tumours. To further increase the vector
Sialic-acid binding immunoglobulin-like lectins
Little is known about the nature and the tissue
safety additional measures to restrict viral replica-
(Siglecs) are a recently described family of car-
distribution of physiological Siglec ligands. Impor-
tion in the CNS are needed.
bohydrate-binding receptors involved in immune
tantly, we identified physiological ligands for both
We have generated miRNA-targeted SFV vectors
regulation. In humans, Siglecs are predominantly
Siglec-7 and Siglec-9 at variable, but significant
by inserting target elements for neuron-specific
expressed on hematopoietic cells, however, with a
levels on a large number of human tumor cell lines
miRNAs into the SFV genome. Results indicate that
highly cell-type specific distribution for each Siglec
from different histological types as well as in tissue
neurovirulent SFV clone carrying target elements
family member.
sections from patients with malignant melanoma,
for miR124 has significantly attenuated replication
We report significant expression of Siglec-7 and
but not in normal melanocytes.
potency in the CNS of adult Balb/c mice while re-
Siglec-9 on human cord and peripheral blood
Altogether, our phenotypic and functional data
taining oncolytic properties tested in vitro.
natural killer (NK) cells. The large majority of
suggest a possible role of Siglec-7 and Siglec-9
Intracranial infection of normal adult Balb/c mice
human NK cells express high levels of Siglec-7;
and their ligand(s) in the inhibition of anti-tumor
with SFV4-miR122T virus carrying liver-specific
in contrast, Siglec-9 is present on a subset of CD-
immune responses. Knowing the importance of NK
target resulted in lethal neurological symptoms in
56dimCD16pos NK cells, while it is absent on CD-
cells in anti-tumor immunity, we believe that our ob-
100% of mice similar to WT SFV4. In mice infected
56brightCD16neg/dim NK cells.
servations might translate in a near future into novel
with neuronal miR124-targeted virus, a statisti-
Interestingly, NK cells isolated from peripheral
therapeutic strategies for treatment of malignancies.
cally significant survival benefit over the SFV4-
blood from patients with malignant melanoma and
miR122 infected group was obtained. When infect-
colon adenocarcinoma display significantly lower
ed i.p. with SFV-miR124T, seven out of eight mice
ex vivo Siglec-9 expression as compared to healthy
remained asymptomatic or showed mild symptoms
donors, arguing for a possible implication of Siglec-
and only weak CNS spreading. Interestingly, i.c.
9 in modulation of anti-tumor immunity.
administered SFV4-miR124T virus was found to
Functionally, in vitro ligation of both Siglec-7 and
infect preferentially the white matter tract of corpus
Siglec-9 results in significant inhibition of NK cell
callosum as reported recently for A774 intracranial
cytotoxicity (e.g. targeT-cell killing, CD107a up-
infection. Asymptomatic mice from SFV4-miR124T
regulation) and engagement of Siglec-7 also sig-
infected group were immune against lethal chal-
nificantly decreases cytokine secretion (e.g. IFNγ).
lenge with SFV strain L10. The results hold promise
Remarkably, in vivo experiments in huNSG mice
for development of safer alphaviral vectors for CNS
confirm the in vitro functional findings.
177
125 | New Targets & New Leads
126 | New Targets & New Leads
MELOE-1 contains multiple HLA class II T-cell epitopes eliciting
Th1 responses in melanoma patients
Identification of hematopoietic minor H antigens using
­leukemia reactive T-cells and genetic linkage analysis
1, 2, 3
1, 2, 3, 4
1, 2, 3
Mathilde Bobinet , Virginie Vignard , Anne Rogel 1, 2, 3, 4
1, 2, 3
1, 2, 3
Dreno , François Lang
, Nathalie Labarriere 1, 2, 3, 4
, Amir Khammari , Brigitte
1
2
1
1
1
Boidinh Chung-Ueck , John Castle , Jana Albrecht , Michaela Frey , Ralf-Holger Voss , Eva
1
1
Distler , Wolfgang Herr 1
Inserm, U892, Nantes, F-44000, France
1
3 Department of Medicine, University Medical Center, Mainz, Germany
2
Univ Nantes, Nantes, F-44000, France
2
Institute of Translational Oncology (TRON) gGmbH, University Medical Center, Mainz, Germany
3
CNRS, UMR 6299, Nantes, F-44000, France
4
CHU Nantes, Nantes, F-44000, France
MELOE-1 is a melanoma antigen encoded by a mes-
ited CD4 responses against at least 1 out of 4 over-
senger overexpressed in melanomas. We previously
lapping peptides. Interestingly, the central region
+
identified a CD8 T-cell epitope from this antigen,
11-30 appears especially immunogenic, with CD4
MELOE-136-44, presented in the HLA-A*0201 context.
specific responses detected in each tested donor.
We reported a strong correlation between the infu-
Responses against this central region are signifi-
The success of an effective treatment of patients
with EBV-transformed B lymphoblastoid cell lines
sion of tumour infiltrating lymphocytes containing
cantly more frequently detected than responses
with leukemia by allogeneic hematopoietic stem
(B-LCL), but not with fibroblasts of patient origin,
MELOE-1 specific CD8 T-cells and relapse preven-
against C-terminus and N-terminus regions, re-
cell transplantation (allo-HSCT) depends on the
suggesting that the target antigens may not be
tion of stage III melanoma patients. Furthermore, a
spectively occurring in 4/6 and 5/6 donors. On the
graft-versus-leukemia (GvL) effect mainly mediat-
broadly expressed mHag. We have analyzed the
large T-cell repertoire against this epitope is present
contrary, the region 18-37 seems poorly immuno-
ed by donor T-cells. Unfortunately, this GvL effect
recognition pattern of two CTL clones (2H9: HLA-
in HLA-A*0201 healthy subjects and melanoma
genic, both in terms of frequency and magnitude
is frequently associated with broad alloreactivity
B*07:02 restricted; 4B2: HLA-C*07:01 restricted) to
patients. Our objective was to define the immuno-
of specific responses. We confirmed these results
of T cells to epithelial organs clinically known as
a panel of previously genotyped B-LCL that express
genicity of this new melanoma antigen, in terms of
in a panel of melanoma patients, and documented
graft-versus-host disease (GvHD). Furthermore, the
HLA-B*07:02 and HLA-C*07:01, respectively, either
class II epitopes, in order to design a therapeutic
that MELOE-1 specific CD4 responses, occurring
desired GvL effect can be sometimes too weak re-
endogenously or after retroviral transduction.
vaccine. We previously indentified two class II epi-
in each patient, were Th1 responses, favourable to
sulting in leukemia relapse after transplantation.
B-LCLs originated from three large pedigrees from
topes, in the vicinity of the HLA-A2 epitope, located
the amplification of CD8 specific T-cells. In order
We see a promising strategy to overcome these lim-
the CEPH reference family collection of the Corielle
in the C-terminus region of MELOE-1. This antigen
to formally identify some of these new class II epit-
itations by adoptive transfer of donor T-cells that
Institute in New Jersey/USA (Pedigree 1332, 1362
being a small protein of 46 amino acids, the use of
opes, we derived CD4 T-cell clones specific for each
specifically recognize leukemia cells as targets.
and 1413 with 50 individuals in total). Based on
this whole polypeptide for vaccination would be
region of the protein. These new epitopes are thus
Minor histocompatibility antigens (mHag), which
this recognition pattern for each CTL clone we have
an interesting option, provided that additional and
located in the N-term region (2-21), presented in the
are polymorphic peptides presented by HLA mol-
divided all 50 individuals into two different groups
immunogenic epitopes could be identified all along
HLA-DQβ1*0202 context, in the central region (11-
ecules, play a crucial role as targets for GvL and
being either mHag-positive or mHag-negative, re-
MELOE-1 sequence.
30), presented by the DRβ1*1101 and the DRß1*0101
GvH reactive donor T-cells. Therefore the knowl-
spectively. We currently perform genetic linkage
Thus, in this study we evaluated the immunoge-
molecules and in the C-term region (26-46), pre-
edge of mHag with specific or at least preferential
analysis using genotypes (microsatellites) of these
nicity of MELOE-1 by stimulating healthy donors
sented in the HLA-DQβ1*0201 context.
expression in hematopoietic tissue would most
individuals, which are online available at the CEPH
and patients PBMC with 10 µM of MELOE-1 whole
We showed that these new epitopes could be effi-
likely allow to therapeutically induce selective GvL
database, to identify linked regions and to narrow
polypeptide, during 14 days. Microcultures were
ciently presented to CD4 T-cells by dendritic cells
responses in the absence of GvHD.
down the region for the final gene identification
then re-challenged with four 20-mer peptides (2-21,
loaded with MELOE-1 whole protein, thus demon-
For the molecular identification of human hema-
11-30, 18-37 and 26-46) corresponding to the four
strating that these class II epitopes can be naturally
topoiesis-specific mHag recognized by CD8 cyto-
future immunotherapy trials are those mHag that
overlapping regions of MELOE-1, followed by cyto-
processed.
toxic T lymphocyte (CTL) clones, genetic linkage
are expressed specifically or at least preferentially
kine labelling of CD4 T-cells.
Thus, we could expect that vaccination of HLA-A2
analysis can be used. We have isolated leukemia-
in the hematopoietic lineage.
We first showed that stimulation of PBMC from
melanoma patients with MELOE-1 whole polypep-
reactive CTL clones by in vitro stimulation of naïve
healthy subjects with MELOE-1 revealed CD4 re-
tide would induce Th1 CD4 responses, stimulating
CD8 T-cells from healthy individuals with fully
sponses specific for the various regions of the protein
the amplification of CD8 effector cells, reactive
HLA class I-matched primary acute myeloid leuke-
in multiple HLA contexts. Indeed, all donors exhib-
against melanoma cells.
mia (AML) blasts. Several CTL clones cross-react
+
+
178
rd
+
+
coding the mHag peptides. Of particular interest for
+
179
127 | New Targets & New Leads
128 | New Targets & New Leads
Identification of unique colorectal cancer T-cell antigens by
next generation sequencing of somatically mutated genes
Identification of lung cancer associated oncoantigens as targets
for active immunotherapy
1
2
1
3
1
Daniele Mennonna , Cristina Maccalli , Michele C. Romano , Renata Bordoni , Gianluca
3
4
4
5
1
De Bellis , Massimiliano Bissolati , Elena Orsenigo , Luca Albarello , Giulia Casorati ,
2
1
Giorgio Parmiani , Paolo Dellabona 1
Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious
­Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy
2
Unit of Immuno-Biotherapy of Melanoma and Solid Tumors, San Raffaele Scientific Institute,
Milano, Italy
3
Institute for Biomedical Technologies, National Research Council, Milano, Italy
4
Unit of Gastro-Enterological Surgery, Department of Oncology, San Raffaele Scientific
Institute, Milano, Italy
5
Department of Pathology, San Raffaele Scientific Institute, Milano, Italy
180
1
1
1
Federica Riccardo , Elena Quaglino , Maddalena Arigoni , Elisabetta Ercole , Manuela
2
3
1
1
1
Iezzi , Dario Livio Longo , Guido Forni , Raffaele Calogero and Federica Cavallo 1
Department of Clinical and Biological Sciences, Molecular Biotechnology Center, University of
Turin, 10126 Turin, Italy
2
Department of oncology and Neurosciences, G. d’Annunzio University, 66100 Chieti, Italy
3
Department of Chemistry IFM and Center for Molecular Imaging, University of Turin, 10126
Turin, Italy
Immunotherapy is a promising strategy to selective-
ated also paired CSCs. A pool of transcripts for
Non small cell lung cancer (NSCLC) is the leading
sponse class and green cluster was characterized
ly attack cancer. The molecular definition of Tumor
20 CAN-genes (described by Wood et al., Science
cause of cancer death in men and women. There-
by the presence of 44 transcripts associated to in-
Associated Antigens (TAAs) recognized by autolo-
2007) has been amplified and subjected to high
fore, the development of new therapeutic strate-
flammation disorder class, agreeing with the con-
gous T-cells has been crucial to develop rationally
throughput sequencing, confirming the presence
gies is essential for improving the prognosis and
solidated link existing between chronic immune
designed clinical protocols. Vaccination strategies
of several somatic mutations in a subset of these
treatment of patients. An interesting experimental
activation and tumorigenesis in human NSCLC.
have concentrated up to now on the use the non-
genes. Synthetic 15-mer peptides spanning the
model to study human lung adenocarcinoma is rep-
Among the genes found differentially expressed in
mutated differentiation or cancer/testis Tumor As-
mutated proteins encoded by every mutated gene
resented by p53
mice. They develop
mouse model and ranked to full-fill the minimal
sociated Antigens (TAAs) as vaccines, because they
expressed by each CRC cell line are being tested for
aggressive NSCLC that metastasize to multiple
requirement for an oncoantigen, CXCR1, SLC16A6,
are conveniently shared by different cancers and/or
their ability to elicit an in vitro immune response.
sites and display a stepwise, directly age-related
SLC26A9 and ROS1 were identified as potential
patients. However, the clinical results obtained as
Preliminary results obtained in one CRC sample
progression, mimicking several features observed
candidate for the building of anti-tumor vaccines
yet with these TAAs showed only marginal clinical
(CRC 1869) show that the frameshift mutation oc-
in lung cancer patients. By using non-invasive mag-
against NSCLC. On the basis of the literature and
benefit. The possible reasons reside in the poor im-
curring in APC generates a neo-epitope that is pre-
netic resonance imaging (MRI), histopathological
mouse data we consider ROS1 an interesting puta-
munogenicity of self shared TAAs, due to the low af-
sented by HLA-DR molecules in tumor cells and it
and immunohistochemical analysis, lung cancer
tive oncoantigen to be further investigated. More-
+
R172H∆g
/Kras
R172H∆g
G12D
finity T-cell recognition, together with their capacity
is recognized by specific CD4 T-cells In addition,
progression in p53
mice was charac-
over, in order to identify novel fusion transcripts
to trigger immune escape mechanisms counteract-
peptides corresponding to the non-synonymous
terized. A significant gender difference in tumor
associated to NSCLC, potentially useful as diag-
ing the anti-tumor effects induced by vaccination.
point mutations in TP53 and SMAD4 elicit CD8
+
progression was observed, with females developing
nostic and therapeutic targets, RNA-Seq studies are
Evidence from animal models and a limited number
and CD4 T-cells that specifically react against the
more tumors than males. Since, the identification
now in progress.
of patients suggest that unique TAAs, encoded by
cancer cells expressing the mutated genes. Alto-
of oncoantigens expressed during tumor develop-
somatically mutated cancer genes, generate strong-
gether, these preliminary results support the fea-
ment could provide an unprecedented opportunity
ly immunogenic epitopes that induce tumor control
sibility of the approach to identify unique TAAs in
to address the immune system against these mol-
by the host’s immune system. We are using high-
CRC by second generation massive sequencing and
ecules, total RNA was extracted from lungs of 10,
throughput DNA sequencing of somatic mutations
reverse immunology. We expect that the substan-
20 and 30 week-old wild type (wt) and p53
+
/Kras
G12D
R172H∆g
G12D
/­
mice. Transcription profiling was per-
in colorectal cancer cells (CRC), and possibly also
tial increase in the number of somatically mutated
Kras
in their Cancer Stem Cells (CSCs), to identify patient-
proteins provided by the planned whole exome se-
formed using Mouse Exon 1.0 ST arrays. 282 genes
specific unique TAAs that can be potentially used in
quencing will markedly increase the likelihood to
were found significantly differentially expressed
the clinics to elicit a strong and specific anti-tumor
identify unique TAAs expressed by CRC cell and
during the increment of the tumor mass. Interest-
immune response.
their CSC derivative in each patient.
ingly, within the top biological functions detected
We have established cells lines from 8 tumor
by IPA, red cluster was characterized by the pres-
specimens of CRC patients, two of which gener-
ence of 10 transcripts related to inflammatory re181
129 | New Targets & New Leads
130 | New Targets & New Leads
Designer host defense peptides for treatment of colorectal
­carcinoma
Characterization of CD4+ T-cell responses specific for novel
HLA-DR-restricted epitopes derived from the breast tumor
­antigen NY-BR-1
1
1
1
1
Claudia Maletzki , Ulrike Klier , Samuel Marinkovic , Jörg Andrä², Michael Linnebacher 1
Section of Molecular Oncology and Immunotherapy, University of Rostock
²
Department of Biochemistry and Molecular Biology, University of Hamburg; Germany
1
1
2
2
1
Translational Immunology, German Cancer Research Center, Heidelberg, Germany
2
Dept. of Medical Oncology, National Center for Tumor Diseases, University Hospital, Heidelberg,
Germany
The emerging resistance towards standard che-
positive control and an irrelevant peptide (NK11)
Breast cancer resembles the most common malig-
tigenic peptide, but also when expressing the target
motherapeutic drugs makes identification and de-
served as negative control.
nant cancer type (26%) of women in the western
antigen endogenously, showing that the epitopes
velopment of new therapeutics imperative. Host
CRC lines harboring high levels of PS were sub-
world with a five year survival rate below 20% for
identified in HLAtg mice were actually represent-
defense peptides are antimicrobial peptides which
stantially impacted by NK-2, even at low concen-
patients with metastasized breast cancer types.
ing naturally processed products in the human
constitute effector molecules of the innate immune
trations. These effects could be attributed to direct
Therefore, innovative therapy strategies that would
system. The frequencies of epitope specific CD4
system. Besides their capacity to act against micro-
cytotoxicity, since viability as determined by
allow efficient treatment of patients with advanced
T-cells among PBMC of patients and healthy donors
bia and fungi, they have surprisingly been found
calcein-AM fluorescence, dramatically decreased
breast cancer are urgently needed. Immunotherapy
will now be determined and the specific reactiv-
to exhibit broad antitumoral activity. The peptide
following NK-2 exposure (IC50: 5 µM). Comparable
approaches, in particular adoptive T-cell transfer,
ity of these T-cells will be tested on human tumor
NK-2, derived from the cationic core region of NK-
effects were obtained for the positive control and,
might represent an attractive strategy to achieve this
cell lines. The use of NY-BR-1-specific CD4 T-cell
lysin, is one of the most promising candidates. The
of particular interest, the derivative 1, but not for
goal. The differentiation antigen NY-BR-1 was found
epitopes might open the possibility for the design
tumor selectivity of NK-2 was found to be (at least in
the derivative 2. Hence, specific tumor cell impair-
to be expressed in breast tissue, testis, prostate, and
of combined immunotherapy approaches based on
part) due to differences in negatively charged mem-
ment may be anticipated. Besides, CRC lines ex-
most notably in 100% of tested breast cancers in situ.
adoptive transfer of tumor antigen specific CD4
brane components, like phosphatidylserine (PS) in
pressing lower levels of PS were also affected by
NY-BR-1 can thus be considered as a suitable target
T-cells sustaining the cytotoxic effector function of
tumor targeT-cells. This is a very unique mode of
the respective peptides. We therefore observed
antigen for T-cell based immunotherapy approaches
co-administered tumor antigen specific CD8 CTL.
action, resulting in rapid cell membrane damage by
only a minor correlation between PS-expression
+
against breast cancer. Tumor antigen-specific CD4
forming lesions and pores in cancer cells. There-
and response rate to NK-2. However, the selectivity
T-cell responses were shown to be essential for ef-
fore, the aim of this study was to comprehensively
of NK-2 towards tumor cells was verified, as we
ficient tumor eradication in various tumor models
analyze the cytolytic potential of NK-2 as well as
did not detect any cytotoxic or hemolytic activity
and in clinical settings. In this project , HLA-trans-
derivatives thereof on patient-derived freshly estab-
towards non-malignant lymphocytes and erythro-
genic (tg) mouse strains expressing HLA-DRB1*0301
lished colorectal carcinoma (CRC) cell lines.
cytes, respectively.
(DR3tg) or HLA-DRB1*0401 (DR4tg) were im-
Generally, freshly established CRC lines exhibited
These promising data underline the high poten-
munized with the NY-BR-1- encoding expression
a heterogeneous expression profile of cell-surface
tial of host defense peptides in general and NK-2
plasmid pcDNA3.1-NY-BR-1 followed by ex vivo
PS. When comparing with non-malignant controls
in special for oncolytic treatment strategies. Sub-
analyses of the NY-BR-1-specific T-cell responses
(B-cell lines, peripheral blood mononuclear cells),
sequent studies will show whether resistance de-
with a synthetic peptide library covering the entire
all CRC cell lines had higher PS levels. However,
velops after long term treatment schedules or if
NY-BR-1 protein. Applying this strategy, a series of
PS levels were lower in the primary cell lines than
the tumor cells remain susceptible towards NK-
HLA-DRB1*0301 and -DRB*0401 restricted epitopes
standard CRC cells (average mean fluorescence in-
2-mediated cell lysis. This will provide the basis
were identified and HLA-DR-restricted CD4 T-cell
tensity: 20 vs. 40).
for preclinical studies in xenopatients to determine
lines specific for the new NY-BR-1-derived epitopes
In subsequent functional analysis, we determined
the effectiveness of host defense peptides against
were established. These murine CD4 T-cell lines
the impact of NK-2 and its derivatives on cell pro-
solid cancers in vivo.
will be analyzed for the recognition of human HLA-
liferation and cytotoxicity. Melletin was used as a
182
1
Adriane Gardyan , Wolfram Osen , Maria Jesiak , Inka Zörnig , Dirk Jäger ,
1
Stefan B. Eichmüller +
+
+
+
+
+
matched targeT-cells not only when loaded with an183
131 | New Targets & New Leads
132 | New Targets & New Leads
Epitopes derived from the mutated region of Nucleophosmine 1
(NPM1) induce both CD4+ and CD8+ T-cell responses
CD4+ T-cells Recognising Human B Lymphoma-Associated
­Antigens
1
1
1
1
1
1
Jochen Greiner , Yoko Ono , Susanne Hofmann , Vanessa Schneider , Anita Schmitt ,
1
1
1
1
1
Lu Zhang , Elmar Mehring , Marlies Götz , Konstanze Döhner , Joannis Mytilineos ,
2
1
1
Markus Wiesneth , Hartmut Döhner , Michael Schmitt 1
Department of Internal Medicine III, University of Ulm, Ulm, Germany
2
Institute for Transfusion Medicine, University of Ulm, and Institute for Clinical
Transfusion Medicine and Immunogenetics GmbH, Ulm, Germany
1
1
School of Cancer Sciences, University of Birmingham, Birmingham, UK
2
School of Immunity and Infection, University of Birmingham, Birmingham, UK
mutated protein showed specific T-cell responses
Epstein-Barr virus (EBV)-specific T-cell prepara-
one of the most frequent molecular alterations and
in healthy volunteers and AML patients. In NPM1-
tions, generated by stimulating immune donor
predominantly occur in AML with normal cytoge-
mutated AML patients 33% showed immune re-
lymphocytes with the autologous virus-trans-
netics. Patients with NPM1 mutation without FLT3-
sponses of CD8 T-cells against peptide P3 and 42%
formed B lymphoblastoid cell line (LCL) in vitro,
ITD mutation show a favourable prognosis of their
against peptide P#3. Specific lysis was detected in
are successfully used to target EBV-positive ma-
disease. The functional role of mutated NPM1 for
chromium release assays NPM1 peptide-primed ef-
lignancies. Whilst these preparations are enriched
the improved clinical outcome is under evaluation.
fector T-cells generated from NPM1-mutated AML
for EBV antigen-specific CD8
Immune responses might be involved in the clinical
patients. Tetramer assays showed peptide-specific
+
contain a CD4 T-cell population whose specificity
outcome of the disease. In this work, we demon-
T-cells. NPM1-peptide-A overlapping MHC class II
is unknown.
strate both CD4 and CD8 T-cell responses against
epitopes were identified by primary structure anal-
Here we show that stimulation of peripheral blood
epitopes derived from the mutated region of NPM1.
ysis program to analyze the role of CD4 T-cells.
lymphocytes with autologous LCL activates not
The entire amino acid sequences of the NPM1 wild
Eight favourable overlapping peptides OL 1 - 8
only EBV-specific CD4 and CD8 T-cell memory,
type protein as well as of the mutated cytoplas-
were synthesized and exploited for CD4
mic NPM1 types A, B, C and D were screened for
stimulation. In granzyme B ELISPOT assays, OL8
HLA-A*0201 binding T-cell epitopes using the al-
co-pulsed NPM1-A CD8 T-cells indicated notable
LCLs and not mitogen (CD40L/IL4)-activated B
gorithms of the SYFPEITHI, the Rankpep and the
S.I., in contrast other OL1-7 disabled to increase
lymphoblasts, they can be isolated from the blood
HLA-Bind software programs. Ten peptides with
granzyme B secretion. To ensure that Th1 cytokine
of EBV-naive as well as –immune donors and
+
the most favourable characteristics were subjected
+
+
+
T-cell
+
+
+
+
+
+
but also a novel set of MHC class II-restricted CD4
effectors. Though these cells likewise recognise
+
appear to recognise not viral proteins, buT-cellular
+
antigens that are up-regulates in EBV-transformed
secretion, under the condition of CD8 and CD4
T-cells mixed culture, resulted from NPM1-A CD8
in 22 healthy volunteers and 27 AML patients to test
T-cells but not HLA-DR epitope stimulated CD4
+
cells. Indeed, a range of EBV-negative B- and Tlymphoma cell lines with appropriate MHC class
specific T-cell responses of CD8 T-cells. Tetramer
T-cells, HLA-A2 blocking effect was confirmed in
assays against the two most interesting epitopes
ELISPOT assay. NPM1-A CD8
T-cells co-pulsed
II types (and more recently at least one lympho-
have been performed and chromium release assays
with OL6, 7 and 8 showed lesser interferon-γ secre-
ma biopsy tested ex vivo) are also recognised by
have been used to show specific lysis by peptide-
tion after HLA-A2 blocking antibody exposure as
such ‘LCL-specific’ effectors, suggesting that these
specific T-cells. Moreover, HLA-DR binding epi-
73, 35 and 57%. Of note, 83-94% of granzyme B
same cellular antigens are frequently up-regulated
topes were screened in algorithmic analysis and
secretion levels were reduced by HLA-A2 blockade
during the process of non-virally-mediated lympho-
HLA-DR*0701 binding peptides were exploited to
+
stimulate CD4 T-cells. In the presence of overlap+
ping peptide stimulated CD4 T-cells, NPM1-A spe+
2
T-cells, most also
to ELISpot analysis for interferon-γ and granzyme B
+
1
1
Mutations in the nucleophosmin 1 (NPM1) gene are
+
184
1
Heather Long , Alison Leese , Nikki Smith , Wenbin Wei , Guy Pratt , Mark Drayson ,­­
1
1
­Martin Rowe , Alan Rickinson +
+
administration, and by which NPM1-A CD8 T-cells
magenesis. Such antigens are therefore of potential
seemed to be the most probable IFN-gamma and
value as targets for CD4
+
granzyme B producers and CD4 T-cells to interfere
+
+
T-cell-based immuno-
therapy. Their identity, means of presentation and
cific CD8 T-cells revealed augmented interferon-γ
with CD8 T-cells.
range of expression in tumour targets are under
and granzyme B secretion and up-regulation of in-
Taken together, mutated NPM1 is a promising
investigation.
tracellular interferon-γ.
target structure for specific immunotherapies in
Two epitopes (P#1 and P#3) derived from the NPM1-
AML patients.
185
133 | New Targets & New Leads
134 | New Targets & New Leads
Novel tumor associated antigens for chronic lymphocytic leukemia
The Quest for Novel Peptide Vaccines in Renal Cell Carcinoma:
Mining the HLA-Ligandome
1, 2
2
2
2
Juliane S. Stickel , Daniel Johannes Kowalewski , Heiko Schuster , Claudia Berlin , Hans2
2
Georg Rammensee , Stefan Stevanovic Daniel Johannes Kowalewski, Armin Rabsteyn, Heiko Schuster, Hans-Georg Rammensee,
Stefan Stevanović
1
University Medical Hospital, Department of Heamatology and Oncology, University of
­Tübingen, Germany
University of Tübingen, Interfaculty Institute for Cell Biology, Department of Immunology
2
Interfaculty Institute for Cell Biology, Department of Immunology, University of Tübingen,
Germany
Chronic lymphocytic leukemia (CLL) is the most
Additionally HLA quantification experiments on
Renal cell carcinoma (RCC) is among the ten most
cation of veritable tumor specific peptides.
common incurable leukemia in western countries.
the cell surface of CLL cells and autologous healthy
common cancers in men in the US. With the esti-
So far we were able to identify more than 7000
Although several novel treatment approaches have
B cells were performed using a flow cytometric in-
mated number of new cases exceeding 60.000 and
peptides predominantly derived from self proteins
been made in recent years mostly showing high
direct immunofluorescence assay.
more than 13.000 RCC-related deaths projected for
with no apparent tumor association. Employing
initial response rates, none of these approaches was
The quantitative results show similar surface ex-
2012, RCC remains a disease with a dire prognosis.
the strategies described above we could highlight
able to extend overall survival.
pression of HLA class I and II molecules on CLL
Approximately 30 % of persons affected present
10 proteins represented by 25 different peptides
Results of bone marrow transplantation as well as
cells compared to autologous normal B lympho-
with metastatic disease, which decreases the
as being overrepresented in the HLA ligandomes
remission phenomena after viral infections suggest
cytes.
5-year survival rate to ten percent. This indicates
of malignant tissues. Furthermore we eluted and
that chronic lymphocytic leukemia might be tar-
We were able to identify a total of more than 2500
an inadequate therapeutic situation which calls for
identified novel peptide ligands from previously
geted effectively by T-cell based immunotherapy.
peptides from 8 CLL patients of all states of disease.
novel/ additional treatment strategies. With RCC
described RCC-associated antigens.
For this goal the identification of tumor associated
The majority of identified peptides are derived
being an immunogenic tumor it is well suited for
Potential TUMAPs were verified by LC/MS-based
HLA (human leukocyte antigen) presented pep-
from self proteins with no apparent leukemia asso-
an immunotherapeutic approach.
peptide sequencing of their synthetic counterparts
tides, which are able to induce a tumor specific cy-
ciation. However we identified several new ligands
In order to develop an efficient peptide vaccine
and are going to be checked for immunogenicity in
totoxic T lymphocyte response, is indispensable. In
derived from established leukemia-associated an-
based immunotherapy of RCC we are directly
future immunological assays. The long-term objec-
comparison to other malignancies only few tumor
tigens (e.g. Fibromodulin), from proteins showing
analyzing the HLA-ligandomes of patient-derived
tive is to implement TUMAPs holding up to these
associated antigens for CLL are described. Thus the
an overexpression on mRNA level in CLL (e.g. SET
tissue samples by LC/MS-based peptide sequenc-
tests in clinical trials with the aim to eventually
aim of this study was to identify novel tumor as-
proto-oncogene) as well as from new, potentially
ing. The obtained data are mined for tumor as-
establish them as off-the-shelf vaccines for the im-
sociated antigens by the first direct isolation and
CLL associated peptides (LEUMAPs) identified by
sociated peptides (TUMAPs) employing various
munotherapy of RCC.
analysis of HLA class I ligands from the cell surface
cross checking all the obtained peptide sequences
strategies such as side by side comparison of the
of CLL patients.
with our internal peptide database for occurrence
HLA-ligandomes derived from bulk tumor with the
Peripheral blood mononuclear cells (PBMC) from
on healthy tissues, especially B lymphocytes and
ligandomes derived from autologous benign tissue.
CLL patients were isolated, followed by magnetic
PBMCs. The sequences of identified LEUMAPs
Furthermore a comprehensive comparison of the
cell separation for purification of the malignant
were verified by LC/MS-based peptide sequencing
combined ligandomes highlights proteins over-
clone. HLA class I ligands were isolated using im-
of their synthetic counterparts and will be tested
represented in malignancy derived ligandomes.
munoprecipitation. Liquid chromatography/mass
for immunogenicity by immunological assays.
This enables the identification of novel RCC associ-
spectrometry (LC/MS) based peptide sequencing
This study paves the way for the development of
ated antigens solely based on the immunologically
was used for the identification of HLA presented
future peptide based immunotherapy of CLL by
pivotal hallmark of HLA restricted presentation
peptides. The obtained data were mined for leuke-
confirming that there is no loss or downregulation
while ostracizing formerly employed criteria such
mia associated peptides by comparison of the HLA
of HLA on CLL cells and by providing a number of
as mRNA and protein expression levels. Finally,
ligandomes of malignant CLL cells with the ligan-
leukemia associated antigens, which for the first
the data were mined for peptides derived from de-
domes of PMBCs and B cells from healthy donors,
time were directly obtained from the HLA ligan-
scribed RCC associated mutations as comprised in
investigation of gene expression databases and lit-
domes of CLL patients.
the COSMIC database (http://www.sanger.ac.uk/
erature research.
186
genetics/CGP/cosmic/), thus enabling the identifi187
135 | New Targets & New Leads
136 | New Targets & New Leads
The HLA class I ligandome of prostate cancer: New targets for
peptide-based immunotherapy
Lymphoma specific immunity in healthy individuals and patients targets phosphorylated antigens derived from the cytoplasmic tail of CD19
1
1
1
1
1*
1*
1
1
1
Christian Hotz , Felix Dingler , Stefan Stevanović , Hans-Georg Rammensee , Arnulf
2
2
Stenzl , Jörg Hennenlotter
Hugo De La Peña , Richard Buka , Thomas Butler , James E. Turner , Sarah Penny , Da1
1
2
1
vid George Millar , Oliver Goodyear , Guy Pratt , Mark Cobbold 1
University of Tübingen, Institute for Cell Biology, Department of Immunology
1
MRC Centre for Immune Regulation, University of Birmingham, Birmingham UK;
Department of Urology, University Hospital Tübingen
2
Heart of England NHS Trust, Birmingham, United Kingdom
2
188
Prostate cancer (PrCa) is highly prevalent. It is the
identified by HLA immunoprecipitation and sub-
Haematological malignancies are unique in their
Furthermore, incubation of circulating lympho-
most common noncutanous cancer and second
sequent peptide elution followed by high-perfor-
susceptibility to curative immunological therapies
cytes with autologous LCLs or HLA-matched
leading cause of cancer deaths among men in the
mance liquid chromatography-mass spectrometry
such as stem cell transplantation, donor lymphocyte
primary Chronic Lymphocytic Leukaemia (CLL)
USA, with estimated 217,730 new cases and 32,050
(HPLC-MS) based peptide sequencing. The result-
infusion and the manipulation of endogenous im-
cells leads to proliferation and expansion of pCD19-
deaths in 2011. The mortality rates for PrCa have
ing peptides, respectively their source proteins, are
munity through immunostimulatory cytokines. The
specific T-cells yet incubation with normal, healthy
been decreasing in many developed countries (e.g.
subsequently screened for their potential tumor
clinical utility of cellular immunotherapies provides
B-cells does not. Moreover, these expanded T-cells
USA, UK, Germany), which has been attributed to
association. Further verification of the tumor-as-
powerful and direct evidence that the human adap-
are effective killers of both autologous LCLs and
improved treatment and early diagnosis. Due to im-
sociated peptides is achieved by comparing gene
tive immune response is able to control and eradi-
primary CLL cells.
provements in detection of prostate specific antigen
expression profiles from different tumor stages and
cate tumours. However, the nature of the antigens
In a mouse model of adoptive immunotherapy, we
(PSA), prostate cancer is detected at an earlier and
different tissues. By this approach we were able to
which are targeted by these effective cellular immu-
have demonstrated in vivo killing capacity of adop-
clinically localized stage, in which curative treat-
identify more than 1700 naturally processed HLA
notherapeutic strategies remains largely undefined.
tively transferred human pCD19 T-cells against
ments could be performed with much better results.
ligands from different HLA allotypes. Most pep-
Cancer genome and epigenetic studies have revealed
lymphoma cell lines after a single dose.
The majority of patients diagnosed with localized
tides derived from self proteins with no apparent
the importance of signal-transduction pathway de-
More recently we have assessed the functional an-
PrCa are successfully treated with radical prosta-
tumor association. Additionally we were able to
regulation in the pathogenesis of cancer. Further-
ti-pCD19 immunity in 17 patients with CLL. This
tectomy or radiation therapy. However, most of the
identify several known prostate-specific antigens
more, therapeutic strategies targeting these have
pCD19-specific immunity is highly heterogeneous
patients will experience a biochemical relapse of
as well as novel candidate tumor antigens. Our
met with considerable success in the clinic provid-
with some patients demonstrating absent immu-
PSA, indicating a hidden local recurrence of the
strategy to verify and validate the tumor-associated
ing a powerful argument for aligning immunother-
nity whilst others have large responses which far
tumor burden. So there is a need for novel thera-
peptides includes comparison by measurement of
apeutic approaches to target signal transduction.
exceed even viral responses.
peutic approaches. Immunotherapy, which boosts
synthetic peptides as well as in vitro cytototoxic
The discovery that phosphorylation of serine and
These data show, for the first time, that both
the patient´s immune response to tumor antigens
T-cell priming assays and finally the application
threonine residues is preserved during MHC class-I
healthy individuals and patients with CLL have
(TA), represents a promising treatment option. In
and the monitoring of immunogenic properties in
and class-II antigen processing pathways suggests
T-cells which exhibit cytotoxicity against primary
order to develop an efficient peptide based immu-
clinical trials.
that phosphopeptide antigens derived from cancer-
CLL cells through recognition of posttranslational-
notherapy for PrCa, it is essential to identify HLA
related phosphoproteins could serve as immuno-
ly modified antigens. In healthy individuals, T-cell
class I presented peptides characteristic for the
logical signatures of ‚transformed self‘.
responses directed against pCD19 may help to
tumor which are able to elicit a specific cytotoxic
Analysis of the peptides displayed on Epstein-Barr
regulate pre-malignanT-cells, thereby revealing a
T-cell response against the malignanT-cells. A pe-
Virus (EBV)-transformed B-cells (LCLs) using a
mechanism of cancer immunosurveillance. These
culiar feature of PrCa are differentiation antigens
mass spectrometry approach, reveals a phosphory-
data increase our understanding of mechanisms
like prostate specific antigen (PSA) or prostatic acid
lated peptide antigen derived from the cytoplasmic
within the adaptive immune response to targeT-
phosphatase (PAP), since the prostate is a nones-
tail of CD19 (pCD19).
cells with underlying deregulated signaling and
sential organ these tissue antigens represent good
Here, we show that CD4 and CD8 pCD19-specific
validate the potential of these phosphopeptides as
targets for immunotherapy of prostate cancer. MHC
T-cells, can be isolated from the peripheral blood
immunotherapeutic targets.
ligands from primary prostate cancer tissue are
of HLA-DRβ1*0101 , HLA-A*0201 healthy donors.
+
+
+
+
189
137 | New Targets & New Leads
138 | New Targets & New Leads
Frequently recognized MHC class II epitopes for an easy generation and detection of EBV specific CD4+ T-cells
High throughput in vitro priming of tumor specific T-cells
1
1
2
1
Christina Kyzirakos , Thomas Feger , Klaus Hamprecht , Hans-Georg Rammensee , Stefan
1
Stevanović 1
Institute of Immunology, University Tübingen, Auf der Morgenstelle, Tübingen, Germany
2
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital, Tübingen, Germany
Stefanie Souczek, Lea Prokop, Kerstin Artzner, Hans-Georg Rammensee, Stefan Stevanović
Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen,
Tübingen, Germany
+
Epstein-Barr Virus (EBV) persists in the vast ma-
flow cytometry. CD4 T-cell clones specific for five
Peptide-based cancer vaccines are one promising
jority of adult individuals (>90%) as a lifelong
epitopes were generated. 24 of the tested peptides
strategy for immunotherapy against cancer. Our
latent infection of the host’s B-lymphocyte pool.
could be identified as MHC class II epitopes by flow
group has characterized large numbers of HLA
Although chronic infections remain asymptomatic
cytometry. A promising mix consisting of five epit-
ligands from tumor cells; among them many that
in most cases, immunocompromised patients can
opes from four different antigens elicited responses
might be used for in vivo induction of tumor-specif-
suffer from severe and life-threatening EBV-associ-
in over 90% of tested donors. A multifunctional
ic immune responses. For the development of pep-
ated diseases, such as post-transplant lymphopro-
CD4 T-cell response could be measured after in
+
+
T-cell
tide-based immunotherapies such peptides should
liferative disorders (PTLD). In immunocompetent
vitro culture with the peptide mix. CD4
individuals the outgrowth of EBV-transformed B
clones specific for four of the five epitopes were able
responses, especially by CD8
cells is successfully controlled by T-cells. Thus,
to recognize autologous EBV infected cells.
a key role in inducing death of tumor cells. The
be able to induce in vivo tumor-directed immune
+
T-cells that have
immunotherapeutic strategies using adoptively
aim of our work is to establish a fast and efficient
transferred EBV-specific T-cells are promising. One
workflow to induce tumor-reactive T-cells which
+
option is the generation and expansion of CD4 and
+
is based on immunogenicity testing of tumor-ex-
T lymphocytes by using EBV-specific syn-
tracted HLA ligands. T-cells usually depend on pro-
thetic peptides for the stimulation of pre-existing
fessional antigen presenting cells (APCs) such as
memory T-cells. Aim of this study was to identify a
autologous dendritic cells (DCs) for specific induc-
set of MHC class II peptides which are recognized
tion and expansion. Since the generation of den-
CD8
+
by specific CD4 T helper cells of basically every
dritic cells is expensive and time consuming with
individual.
varying quality and amount of differentiated DCs,
15 amino acid long candidate epitopes of nine im-
we established an artificial system to replace cell-
munodominant antigens, three of the latent cycle
based in vitro priming of T-cells: Streptavidin-coat-
(EBNA3A, LMP1 and LMP2) and six of the lytic
ed artificial antigen presenting cells loaded with
cycle (BMLF1, BMRF1, BRLF1, BZLF1, BNRF1 and
defined amounts of recombinant HLA molecules
BLLF1) of the virus were predicted using the SY-
and costimulatory antibodies such as anti-CD28
FPEITHI epitope prediction programme (www.
and anti-CD137 (4-1BB). Employing artificial APCs
syfpeithi.de). For each antigen promiscuous pep-
without the need for autologous DCs will lead to
tides with high SYFPEITHI scores were screened
successful in vitro priming of tumor-reactive T-cells
for immunogenicity using IFN-γ-ELISPOT. Each
that might provide an indication for novel peptides
candidate epitope was tested on PBMCs of at least
for immunotherapy.
15 healthy blood donors after in vitro amplification of specific T-cells. Epitopes were confirmed by
190
191
139 | New Targets & New Leads
140 | New Targets & New Leads
Identification of a tumour-associated autoantibody signature
with high diagnostic value for early detection of gastric cancer
Spontaneous humoral antibody responses against tumor-­
associated antigens in malignant melanoma patients
1
1
2
1
1
1*
1*
2
1
1
Karina Silina , Pavel Zayakin , Guntis Ancans , Zane Kalnina , Irena Meistere ,
1
1
1
2
1
Angelina Pismennaya , Diana Andrejeva , Lasma Ivanova , Marcis Leja , Aija Line Inka Zörnig , Niels Halama , Justo Lorenzo Bermejo , Alexander Migdoll , Claudia Ziegelmeier ,
1
3
1
4
3*
1*§
Iris Kaiser , Elke Dickes , Niels Grabe , Christine Falk , Stefan B. Eichmüller and Dirk Jäger 1
Latvian Biomedical Research and Study Centre, Riga, Latvia
1
2
Riga Eastern Clinical University hospital, Latvian Oncology Center; University of Latvia,
­Faculty of Medicine, Riga , Latvia
National Center for Tumor Diseases, Dept. of Medical Oncology, University Medical Center
Heidelberg, Heidelberg, Germany
2
Institute of Medical Biometry and Informatics, University Medical Center Heidelberg and
Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg,
Germany
3
Translational Immunology, German Cancer Research Center, Heidelberg, Germany
4
Immunomonitoring Unit, National Center for Tumor Diseases and Institute for Immunology,
Heidelberg and Integrated Research and Treatment Center Transplantation, Hannover Medical
School, Hannover, Germany
*
contributed equally
§
corresponding author: [email protected]
Introduction: The lack of reliable markers for the
prising serum samples from 235 GC patients, 154
early diagnosis of gastric cancer is a major draw-
peptic ulcer and gastritis patients and 213 healthy
back in managing this disease. Tumour-associated
controls to identify autoantibodies with the highest
autoantibodies are considered to be attractive bio-
diagnostic value.
markers for such purposes due to their specificity
Results: ROC curve analysis showed that a minimal
and stability in sera, however no antibody-based
set of 45 autoantibodies could discriminate GC and
test has been developed so far as each individual
healthy controls of the validation set with an AUC
tumour differs in its antigen repertoire and the
of 0.79 (59% sensitivity and 90% specificity), GC
frequency of autoantibodies against each single
and peptic ulcer with AUC of 0.76, and GC and gas-
antigen is too low (<20%).
tritis with AUC of 0.64. Moreover, it could detect
Methods: We tried to overcome these obstacles by
early GC with equal sensitivity than advanced GC.
(i) applying T7 phage display-based SEREX tech-
Interestingly, the autoantibody production (serum
nique for the identification of a representative set
score) did not correlate with histological type, H.
of antigens eliciting humoral responses in gastric
pylori status, grade, localization and size of the
cancer (GC) patients, by (ii) producing a 1150-
primary tumour while it appeared to be associated
feature phage displayed antigen microarrays and
with the metastatic disease.
exploiting them for the survey of the autoantibody
Conclusions: The identified 45 tumour-associated
repertoire in 100 GC patients of various stages and
autoantibody signature can detect gastric cancer
100 cancer-free controls, and by (iii) developing
with a higher specificity than any other known
specific procedures for antigen microarray data nor-
marker and can be used for the detection of even
malization and cut-off determination of sero-posi-
early stage gastric cancer with equal precision.
tive signals. Each antigen received a rating value
according to the reactive antibody signal intensity
and frequency of reactivity in cancer patients and
healthy donors. Then each serum acquired a value
– the serum score – by summing the ranked signals
of each reactive antigen in that serum, and this
value was used for the statistical analyses of the
autoantibody test. Next, the top-ranked 86 antigens
were used for the production of a focused array that
was tested with an independent validation set com192
Running title: Humoral antibody responses against
responses in all disease stages. Humoral immune
tumor-associated antigens
responses against single antigens were either as-
Purpose: Spontaneous humoral (auto-) immune
sociated with poor prognosis and/or shorter pro-
responses in melanoma patients have been previ-
gression-free survival (PFS) or had no influence.
ously described. However, the distribution, pat-
Among stage IV patients, no significant associa-
terns and prognostic impact of antibody responses
tion between the presence of an antibody response
against specific candidate tumor-associated anti-
and overall survival or PFS was found, whereas in
gens (TAAs) are unknown so far and were inves-
stages I-III significant associations were observed.
tigated in this study for the first time. The final
Multivariate analyses identified specific antigen re-
aim was to identify new prognostic biomarkers for
sponses as prognostic factors independently of age,
malignant melanoma.
chemotherapy and immunotherapy.
Patients and Methods: Two sets of serum samples
Conclusion: Antibody responses against specific
from 465 melanoma patients and healthy controls
TAAs in stage I-III melanoma patients correlate
were investigated. 97 patients with stage I mela-
with poor prognosis and/or shorter PFS. Present
noma, 87 with stage II, 92 with stage III, 89 with
results may help to design clinical studies in order
stage IV and 100 healthy controls were analyzed.
to evaluate the potential of these responses as
Samples were drawn at the time of diagnosis (stage
prognostic serological biomarkers. The humoral
I-III) or at the time of diagnosis of distant metasta-
immune response appears to have detrimental
sis (stage IV). Antibody responses against 26 candi-
effects on the clinical course of the disease.
date TAAs were determined using a novel multiplex
assay and the association between response and
patient survival was investigated.
Results: Antibody responses were heterogeneous
and were mainly found in melanoma patients regarding both frequency and magnitude across different stages. All antigens tested elicited immune
193
141 | New Targets & New Leads
142 | New Targets & New Leads
A peptide microarray platform with a new robust data analysis
package
Human chorionic gonadotropin exerts immunosuppressive
­effects in a mouse model of Graft-versus-Host disease
1
2
1
3
1
Martin Löwer , Bernhard Y Renard , Yvonne Kühne , Ulf Reimer , Andrée Rothermel ,
3
3
3
1
1
Johannes Zerweck , Tobias Knaute , Holger Wenschuh , Özlem Türeci , John Castle , and
1
Ugur Sahin 1
The Institute for Translational Oncology and Immunology (TrOn), 55131 Mainz, Germany.
2
Research Group Bioinformatics (NG 4), Robert Koch-Institute, 13353 Berlin, Germany.
3
JPT Peptide Technologies GmbH, 12489 Berlin, Germany.
Microarrays are versatile tools for answering a
and compared to two existing algorithms. rapmad
plethora of biological questions. DNA microarrays
shows competitive and superior behaviour.
even made their way into clinical diagnostics with
The combination of a flexible peptide array platform
a number of FDA-approved in vitro diagnostic tests.
with our novel and robust data analysis package
The technological development in the field of DNA-
provides an efficient technology for using in differ-
microarrays fertilized other areas as peptide mi-
ent emerging „omics“ as interactomics, kinomics or
croarrays.
acetylomics as well as biomarker discovery.
Peptide Microarrays have been proved useful in many
applications as enzymatic profiling, for instance
of histone modifying enzymes, e.g. kinases and
­deacetylases, or the screening of humoral immune
response towards pathogens or autoantigens.
Here, we present a peptide microarray platform for
the screening of numerous samples on high content
peptide microarrays and a subsequent robust data
evaluation pipline.
Based on a high-throughput peptide synthesis platform, complex peptide libraries are generated and
chemoselectively immobilized onto microarrays in
a density up to 7000 peptides in triplicates.
To achieve a maximum sensitivity we developed
data analysis procedures which are implemented
in the R package rapmad (Robust Alignment of
Peptide MicroArray Data).
The analysis pipeline was evaluated with data from
patient samples on high density peptide arrays
194
Caroline Steinmetz, Steffen Lorenz, Peter Galle, Dennis Strand, Susanne Strand
I
Department of Internal Medicine, University Medical Center, 55101 Mainz, Germany
During pregnancy an immune tolerant micromi-
analysis showed increased phosphorylation of Sirt1
lieu prevents rejection of the alloantigenic embryo.
in the liver of mice treated with hCG. Quantitative
Human chorionic gonadotropin (hCG) may play a
Real Time PCR- and Western Blot analysis revealed
role in helping to establish this situation. Never-
a clear decrease in Bim expression.
theless, the immune response against pathogens is
Based on the immunosuppressive activity of hCG,
maintained. Mimicking this immunological state
it might be a new therapeutic agent for the treat-
might be a desirable strategy to prevent the compli-
ment of GvHD.
cations of Graft-versus-Host disease (GvHD) after
stem cell transplantation.
Using a murine autoimmune hepatitis model we
could show that hCG prevents T-cell mediated liver
damage. The analysis of the signaling pathways
revealed that hCG regulates the longevity protein
Sirt1, which results in an inhibition of the forkhead
transcription factor Foxo3a. This led in turn to a
downregulation of its proapoptotic target gene Bim.
In a GvHD mouse model we report that hCG leads
to a significant reduction of liver damage, indicated
by lowered transaminase values.
Furthermore we performed immuno-stainings for
+
CD4 T-cells in liver and skin sections and found
less infiltration in the hCG-treated mice. This could
be an effect of the attenuated release of IL-16, a
+
chemoattractant factor for CD4 T-cells. Immunostaining of Sirt1 revealed an upregulation of Sirt1 in
the nucleus after hCG treatment. Also Western Blot
195
143 | New Targets & New Leads
Expression and impact of interleukin-22 in human lung cancer
1
1
2
2
Stefanie Völk , Natascha Jennifer Küpper , Till Clauditz , Sarah Minner , Amanda
3
4
5
5
1
1
Tufman , Peter Düwell , Michael Lindner , Ina Koch , Melanie Merk , Simon Rothenfußer ,
4
3
2
2
1
Max Schnurr , Rudolph Maria Huber , Guido Sauter , Waldemar Wilczak , Stefan Endres
1
and Sebastian Kobold
1
Division of Clinical Pharmacology, Department of Internal Medicine IV, Ludwig-Maximilians
Universität München, Munich, Germany
2
Institute for Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
3
Department of Internal Medicine V, Ludwig-Maximilians Universität München, Munich, Germany
4
Department of Internal Medicine IV, Ludwig-Maximilians Universität München, Munich, Germany
5
Asklepios Biobank für Lungenerkrankungen, Asklepios Fachklinik München-Gauting,
Gauting, Germany
Interleukin-22 (IL-22) is an interleukin-10-related
dependent proliferation and cell anabolism but had
cytokine with unique functions in interleukin-
no effect on the migration of these lung cancer cell
22-receptor-1 (IL-22-R1) expressing epithelial cells.
lines. Using annexin V-propidium iodide staining
Recently, it has been suggested that IL-22 may be
and cell titer blue assays (vitality), we found no
an autocrine factor in lung cancer. However, the
rescue from chemotherapy (carboplatin and 5-fluo-
prevalence of this cytokine and its role in the pro-
rouracil) by IL-22 treatment. In contrast, when cells
motion of human lung cancer are not known.
(A549, HCC827) were continuously exposed to cis-
We fi rst screened two cohorts of 205 and 2145 lung
platin until they grew drug-resistant to this drug,
cancer samples for IL-22 expression by immuno-
we found a striking upregulation of the IL-22-R1
histochemistry on a tissue microarray. IL-22 was
both on protein and mRNA level. IL-22 stimulated
detected most frequently in small cell lung cancer
cisplatin-resistanT-cells exhibited higher prolifera-
(n = 50) and large cell lung cancer (n = 303) with
tion rates and higher bcl2 expression than the non-
58 and 46 % respectively. The remaining histo-
resistant controls.
logical types (squamous cell, adenocarcinoma and
Our data suggest that IL-22 expression in tumor
invasive adenocarcinoma with predominant leptic
tissue may not be a prognostic factor in resectable
growth) were significantly less positive for IL-22
lung cancer at the time of diagnosis. In contrast,
(28, 33 and 24 % respectively). IL-22 expression did
our results indicate that in chemotherapy-resistant
not correlate with survival time in any of these sub-
tumor cells, upregulation of IL-22-R-1 and of IL-
types. Next, we addressed why, despite the expres-
22-responsiveness may contribute to more aggres-
sion of IL-22 as a putative protumoral factor, the
sive behavior of the disease.
Therapeutic Vaccination
course of the disease seems unaltered. We analyzed
the effects of IL-22 in five human lung cancer cell
lines (A549, HCC827, H1339, H187 and LOU-NH91).
Expression of IL-22-R1 was determined by Western
blot and quantitative-PCR. IL-22-R1 was expressed
in all analyzed cell lines but the expression level
differed between the cell lines. Elevated levels of
IL-22-R1 were associated with a high response rate
to respond to IL-22 treatment. IL-22 induced STAT3196
197
144 | Therapeutic Vaccination
145 | Therapeutic Vaccination
Tumor cells infected by Measles virus vaccine induce plasmacytoid dendritic cell (pDC) maturation and tumor antigen
­cross-presentation
Use of Oncolytic Rhabdoviruses as Potent Tumour Vaccine
Boosters
*
Jean-Baptiste Guillerme, Nicolas Boisgerault, David Roulois, TANGY Frédéric Tangy ,
­Jean-François Fonteneau and Marc Gregoire
1
2
1
3
1
McMaster Immunology Research Centre, McMaster University, Hamilton, ON, Canada
INSERM U892 – Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Nantes, France
2
Department of Pathobiology, University of Guelph, Guelph, ON, Canada
*
3
Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada
Institut Pasteur PARIS – Unité Génomique virale et Vaccination.
Plasmacytoid Dendritic Cells (pDC) are antigen-
amount of type I IFN and cross-present tumor
Oncolytic viruses are able to mediate tumour de-
alterations to the tumor microenvironment leading
presenting cells specialized in antiviral response.
antigen.
struction in many animal models and some human
to large increases in tumour antigen-specific TILs.
patients but the effects of the viruses are generally
Remarkably these boosting vectors generate larger
quite transient as host immunity ultimately limits
T-cell responses in tumour-bearing hosts than they
viral delivery and spread. However host immunity
can in tumour-free hosts as they replicate in tumour
healthy cells. We have previously demonstrated
can also be beneficial in this context if recruited
cells thus amplifying their boosting capacity.
that myeloid dendritic cells (DC), cocultured with
to attack the tumour during, and importantly fol-
Oncolytic rhabdoviral vectors are extremely potent
MV-infected tumor cells, actively matured and
lowing therapy. If one could turn the transient
vaccine boosters when administered intravenously
cross-presented tumor antigen. Here, we inves-
benefits of viral oncolysis into an ongoing thera-
allowing for systemic delivery to antigen presenting
tigated the effects of MV-tumor-infected cells on
peutic effect by inducing a potent anti-tumoural
cells and tumour beds. These viruses can generate
phenotype and functions of pDC.
immune response the dual effects of viral oncoly-
very large immune responses against both foreign
We compared maturation, cytokine production and
sis plus anti-tumoural immunity may prove highly
and autologous antigens and are able to do so even
tumor-antigen cross-presentation by pDC, exposed
beneficial. Unfortunately, the immunogenic nature
during the peak of the primary response allowing
either to the virus alone, to MV-infected or UV-ir-
of oncolytic viruses means that host immune re-
for rapid generation of potent immune responses
radiated tumor cells.
sponses against viral antigens dominate induced
capable of destroying significant tumour burden in
We found that MV alone or MV-infected cells
responses against bona fide tumour antigens. Thus
aggressive syngeneic tumour models.
induced pDC maturation with a strong IFN-α pro-
novel approaches able to enhance antitumor im-
Our team is developing a novel rhabdoviral platform
duction, whereas UV-irradiated tumor cells did not.
munity during oncolytic therapy may prove highly
based on the Maraba virus. This virus shows ex-
We demonstrate that type I IFN secretion is due to
beneficial.
cellent oncolytic properties in pre-clinical tumour
TLR-7 triggering. We also observed that MV-infect-
We have developed recombinant oncolytic vaccines
models and is a very strong booster of tumour vac-
ed cells were taken up by pDC. Interestingly, we
where we incorporate tumour antigen transgenes
cines. We are currently preparing for a first in man
observed cross-presentation of the tumor antigen,
The measles virus vaccine (MV) was recently proposed in virotherapy as an antitumor agent to target
and specifically kill tumor cells without infecting
This work was supported by INSERM, «Association pour
la Recherche contre le Cancer» (ARC) and «Ligue contre le
cancer inter-région du grand ouest».
into oncolytic viruses to direct the host immune
phase I clinical trial using an MG1 Maraba-MAGE
NYESO-1, to a specific CD8 T-cell clone only when
response toward tumor antigens. In this manner
A3 oncolytic vaccine vector. We will present pre-
pDC were cocultured with MV-infected tumor cells.
we use the oncolytic virus to drive a specific anti-
clinical data from murine tumour model studies as
To our knowledge, we presently demonstrate for
tumoural immune response while performing viral
well as vaccine response and toxicity data from our
the first time the property of tumor antigen cross
oncolysis. In particular, when used in tumor-bear-
ongoing non-human primate trials.
presentation by human pDC.
ing pre-vaccinated hosts, these oncolytic vaccines
Altogether, our results suggest that the use of MV
provide a powerful boost to pre-existing anti-tu-
as an antitumor virotherapy induce immunogenic
mour immunity, while maintaining the benefits of
tumor cell death, allowing pDC to produce large
an oncolytic virus in terms of tumor debulking and
+
198
1
Jonathan Pol , Byram W. Bridle Yonghong Wan , David F. Stojdl and Brian D. Lichty 199
146 | Therapeutic Vaccination
147 | Therapeutic Vaccination
HLA-A*0201+ plasmacytoid dendritic cells provide a cell-based
immunotherapy for melanoma patients
Intravaginal immunostimulation after vaccination increase
­local vaccine-specific CD8 T-cells and tumor regression of
­genital tumors in mice
1, 2
2, 3
2, 4
1, 2
1
1
1
1
Caroline Aspord , Julie Charles , Dimitri Salameire , David Laurin ,
1, 2
2, 3
1, 2
Laurence Chaperot , Marie-Therese Leccia , Joel Plumas Sonia Domingos-Pereira , Loane Decrausaz , Laurent Derré , Martine Bobst , Pedro Rome2
3
1
1
ro , John T.Schiller , Patrice Jichlinski , Denise Nardelli-Haefliger 1
EFS Rhone-Alpes, R&D Laboratory, La Tronche, F-38701 France
1
2
University Joseph Fourier, Grenoble, F-38041 France; INSERM, U823, Immunobiology &
Immuno­therapy of Cancers, La Tronche, F-38706 France
Department of Urology, Centre Hospitalier Universitaire Vaudois and University of Lausanne,
1011 Lausanne, Switzerland
2
Ludwig Center for Cancer Research of the University of Lausanne, 1011 Lausanne, Switzerland.
3
Laboratory of Cellular Oncology, National Cancer Institute, NIH Bethesda, MD 20892, USA
3
CHU Grenoble, Michallon Hospital, Dermatology, pole pluridisciplinaire de medecine, Grenoble, F-38043 France
4
Anatomo-cytopathology department, Michallon Hospital, Grenoble, F-38043 France
Therapeutic options to treat metastatic melanoma
approach provides a pre-clinical basis for the de-
Human papillomaviruses (HPV)-related cervical
tumor regression upon ivag CpG was mediated by
are limited and poorly effective. Yet many evi-
velopment of pDC-based vaccine and an alternative
cancer is the second leading cause of cancer death
CD8 T-cells. Mechanisms of CD8 T-cell recruitment
dences suggest that tumor-specific T-cells have the
tool to produce tumor-specific T-cells for adoptive
in women worldwide. HPV E6 and/or E7 oncogene
in the genital mucosa are currently being exam-
potential to control the tumor. One promising ther-
cellular immunotherapy of melanoma patients.
specific therapeutic vaccines are under active de-
ined. These findings suggest that topical applica-
apeutic option is to induce or transfer melanoma-
velopment, but have had limited clinical efficacy
tion of immunostimulatory molecules might sub-
specific T-cells. Current approaches to elicit such
to date. We hypothesized that specific immune re-
stantially increase the effectiveness of parenterally
T-cells are either not clinically efficient enough or
sponses not only need to be targeted to the tumor
administered vaccines in reducing HPV-induced
fastidious processes that impede their extensive
site but they need to overcome local complex im-
genital neoplasia in women.
clinical use. As pDCs play a crucial role in trig-
munosuppressive tumor microenvironment. We
gering anti-tumor immunity especially in mela-
have developed a new strategy that consists in a
noma, we explored their potential as a cell-based
tumor associated antigen (TAA)-vaccination fol-
approach for melanoma immunotherapy. We inves-
lowed by the use of immunostimulants at the
+
tigated the potency of a human HLA-A*0201 irra-
mucosal site where the tumor reside. Here we
diated pDC line loaded with peptides derived from
report that intravaginal (ivag) administration of
the four major melanoma tumor antigens, MelA/
CpG-ODN (CpG, a TLR9 agonist) or poly (I:C) (PIC,
MART-1, gp100/pmel17, tyrosinase and MAGE-A3,
a TLR3 agonist) was able to increase approximately
to trigger functional multi-specific T-cells ex vivo
5-fold the number of vaccine-specific IFN-γ secret-
+
200
from a large series of HLA-A*0201 stage I-IV mela-
ing CD8 T-cells in the genital mucosa of mice after
noma patients’ PBMC and TIL. The pDCs loaded
subcutaneous (s.c.) E7 vaccination, without affect-
with melanoma-derived peptides promptly induced
ing the E7-specific systemic response. Ivag CpG or
melanoma tumor-specific T-cells from both PBMC
PIC locally increased both E7-specific and total CD8
and TIL of melanoma patients. Responses towards
T-cells, but not CD4 T-cells, most probably through
MelA, gp100, tyrosinase and MAGE-A3 reached tet-
recruitment from the periphery. This previously
ramer levels up to respectively 62%, 41.4%, 85%
unreported recruitment of activated CD8 T-cells by
and 6% in 20 days. The pDC-primed central/effec-
ivag CpG or PIC was mediated by TLR9 and TLR3/
tor memory anti-tumor T-cells are highly function-
Mda5 signaling pathways, respectively. Most inter-
al as they specifically secreted IFNγ and expressed
estingly, ivag CpG after s.c. E7 vaccination of mice
membrane CD107 upon stimulation, and exhibited
bearing large established E7-expressing genital
a strong cytotoxic activity towards semi-allogeneic
tumors resulted in more efficient tumor regression
but also patients’ own melanoma tumor cells. The
than vaccination alone. Antibody-mediated cell-
simple design and potent efficacy of this promising
specific depletion confirmed that this enhanced
201
148 | Therapeutic Vaccination
149 | Therapeutic Vaccination
Characterization of the human CD83 promoter complex for
transcriptional targeting of dendritic cells in vivo
Stimulation of dendritic cells with vaccine and vaccine-­
antibody complex and effect on immune response
1*
1*
2
2
1
1, 2
1
1
1
Ilka Knippertz , Marcello F. Stein , Stefan Lang , Thomas Winkler , Andrea Deinzer ,
1
3
4, 5
1
­Sebastian Erber , Dirk M. Nettelbeck , Thomas Werner , Alexander Steinkasserer Ibrahim Hatipoglu , Duygu Ercan , Ibrahim Sogut , Soner Aksu ,
2
1
Hulya Sivas , Aynur Basalp 1
Department of Immune Modulation at the Department of Dermatology, University Hospital
Erlangen, D-91052 Erlangen, Germany
1
TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute,
P.O. Box 21, 41470 Gebze – Kocaeli, TURKEY
2
Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-AlexanderUniversity Erlangen-Nuremberg, D-91052 Erlangen, Germany
2
Anadolu University, Faculty of Science, Department of Biology, Eskisehir 26470, TURKEY
3
Helmholtz-University Group Oncolytic Adenoviruses at the DKFZ (German Cancer Research
Center) and Department of Dermatology, Heidelberg University Hospital, D-69120 Heidelberg,
Germany
4
c/o Genomatix Software GmbH, D-80335 Munich, Germany
5
Internal Medicine, Nephrology, University of Michigan, SPC5680 Ann Arbor, MI 48109, USA
*
Joint first authorship: These authors contributed equally to this work.
202
Dendritic cells (DCs) are the most important antigen
85 bp downstream of the minimal promoter region
Dendritic cell (DC) vaccine is a promising and potent
presenting cells (APC) since only DCs are able to
which was shown to be the missing link to tightly
therapeutic tool for chronic diseases, autoimmune
induce naive immune responses which makes them
regulate cell-type and stadium-specific human
diseases and cancer because of DC’s unique ability
optimal candidates for immunotherapy of cancer.
CD83 expression. Moreover, these DNA regions
to stimulate T-cells. The challenge of DC vaccine is
One of the most prominently up-regulated mole-
contain a complex framework of IRF- and NFκ B-
to find an effective form for antigen presentation.
cule during the maturation process of DCs is CD83,
transcription factor binding sites mediating their
Although pure antigens, antigen complexes, plas-
thereby representing one of the best-known surface
exact arrangement in DCs. Mutation of any of the
mids and mRNA were used in different studies, any
marker for fully mature DCs. However, the molec-
IRF-binding sites resulted in a significant loss of
proper application to overcome this problem has
ular mechanisms leading to its specific transcrip-
promoter activity, whereas over-expression of NFκ B
not been found yet.
tion are completely unknown. To determine these
transcription factors clearly enhanced transcrip-
In this study, we investigated the eligibility of
mechanisms regulating the cell type- and stadium-
tion. Finally, this highly complementary tripartite
commercial HBV vaccine and vaccine-monoclonal
specific human CD83 expression, ChIP-on-chip-,
promoter-enhancer complex was shown to effi-
antibody complex for antigen loading of DCs for
biocomputational- and gene reporter analyses were
ciently drive therapeutic transgene expression, i.e.
a therapeutic purpose. DCs were derived from the
performed. These studies revealed a transcriptional
of MelanA and IL-12p70, in the DC cell line XS52.
bone marrow of transgenic Hepatitis B (HBV-tg)
activation complex formed by a 3D folding process
Thus, this DC-specific CD83 promoter complex now
mice using GM-CSF, IL-4 and then loaded with a
involving three distinct DNA regions. Such a tripar-
allows for the development of new in vivo target-
commercial HBV vaccine (that contains hepatitis
tite folding process has not been reported for any
ing strategies for next generation DC-vaccination
B virus surface antigens and aluminum hydroxide
other gene so far. By ChIP-on-chip microarray we
directly in patients suffering from cancer.
adjuvant) and vaccine-antibody complex. HBV-tg
could identify a highly transcriptional active region
mice were immunized with vaccine and vaccine-
within the human CD83 gene locus, in mature DCs.
antibody loaded DCs. Optimum HBV vaccine con-
Following deletion mutagenesis, we could charac-
centration and loading time were determined by
terize a short enhancer region of 185 bp. Impor-
WST-1 and BrdU methods. Therapeutic effects of
tantly this regulatory element was not active in im-
vaccine-antibody loaded DCs were determined
mature DCs, which induce tolerance mechanisms,
by the evaluation of antibody response, IFNγ and
or other cells expressing CD83, such as subsets of
HBs expression levels in HBV-tg mice. Our results
activated B- and T-cells. Further biocomputational
showed that commercial HBV vaccine loaded DCs
analyses identified an upstream promoter, located
induced humoral response in HBV-tg mice but it
has no effect on cellular immunity.
Keywords: Dendritic cell vaccine, Hepatitis B virus, HBV
vaccine
203
150 | Therapeutic Vaccination
151 | Therapeutic Vaccination
A pilot study of peptide-based vaccines in combination with
poly ICLC in patients with WHO grade 2 low-grade glioma
Actively personalized multi-peptide vaccination for primary
liver cancers – a novel strategy for overcoming residual disease
1
1
1
1
2
Hideho Okada , Lisa H. Butterfield , Masashi Sakaki , Aki Hoji , Andres M. Salazar ,
3
1
Edward G. Shaw , Frank S. Lieberman
1
University of Pittsburgh Cancer Institute, Pittsburgh, PA 15217, USA
2
Oncovir Inc., Washington, DC 20008, USA
3
Wake Forest Baptist Medical Center, Winston Salem, NC 27157, USA
1, 3
1
3
1, 3
1
University of Tübingen, Institute for Cell Biology, Department of Immunology, Tübingen, Germany
2
Department of Medical Genetics, Institute of Human Genetics, Tübingen, Germany
3
Department of General, Visceral- and Transplant Surgery, University of Tübingen, Gerrmany
Introduction: Adult patients with WHO grade 2
vivin, and WT1. As these GAAs are expressed not
Primary liver cancers, mainly hepatocellular car-
differences between tumor and germline genome
low-grade glioma (LGG) have a significant risk of
only in a subset of LGG cells but even at higher
cinomas (HCC), are among the five most common
are determined and specific databases are gener-
tumor progression despite treatment with surgery
levels in HGG cells, our vaccine approach may offer
cancers globally and a leading cause of cancer
ated. Tumor specific peptides are predicted using
or surgery followed by radiation therapy (RT) and
both immunotherapeutic and immunoprophylactic
related death. More than half a million patients
SYFPEITHI database to facilitate a targeted search
/or chemotherapy, and most patients eventually
potential to reduce the risk of tumor recurrence.
are diagnosed with HCC every year. Especially in
for mutated peptides in the MS -data. In addition
die of the disease. High-risk subsets of these LGG
Results: To date, 13, 1 and 10 patients have been
Europe and the USA incidence numbers have been
to tumor tissue adjacent benign tissue of patients
patients display astrocytoma or oligoastrocytoma
enrolled in Cohorts 1, 2 and 3, respectively. No
rising over the last decades.
was analyzed in the same fashion. So far sample
histology plus any one of the following conditions:
dose-limiting non-CNS toxicity has been encoun-
With chemotherapies and other adjuvant strategies
pairs from seven patients have been investigated
1) age ≥40 with any extent resection; 2) age 18-39
tered except for one case with Grade 3 fever (Cohort
showing only limited benefit, available therapeutic
in this manner.
with incomplete resection; or 3) age 18-39 with neu-
1). ELISPOT assays, completed in 7 and 1 patients
options are scarce. Therefore primary malignan-
Based on this approach, we aim at providing an
rosurgeon-defi ned gross total resection with tumor
in Cohorts 1 and 2, respectively, demonstrated
cies of the liver are a promising entity for targeted
actively personalized vaccine cocktail of individual
size ≥ 4 cm in diameter. These patients have as high
robust and sustained interferon (IFN)-γ (type-1) re-
immune intervention after surgery.
patient- and tumor-specific peptides for adjuvant
as an 89% risk of recurrence by 5 years following
sponses against at least 3 of the GAA epitopes in all
In order to provide a novel effective and targeted
tumor therapy. This approach is completely new
surgery.
cases, while IL-5 (type-2) responses were absent or
adjuvant therapeutic approach, we focus on natu-
and may offer the chance of overcoming residual
Methods: Based on encouraging data from a phase I
transient in all cases. The magnitude of the IFN-γ
rally processed and presented MHC-ligands eluted
disease, which is a major cause of tumor relapse
vaccine study targeting multiple glioma-associated
ELISPOT responses in the current study was sig-
directly from the tumor. Our interest is particular-
so far.
antigen (GAA) epitopes in adult high-grade glioma
nificantly higher than that observed in our previ-
ly focused on mutated peptides representing neo-
(HGG) patients, we initiated a bi-institutional
ous phase I/II study in HGG patients (Okada H et
epitopes derived from specific tumor mutations ex-
pilot study of subcutaneous vaccinations with
al. 2011). Although evaluation of progression-free
clusively present on malignant tissue. Those tumor
synthetic peptides for GAA epitopes emulsified
survival would require a longer observation period,
specific peptides lack central tolerance and should
in Montanide-ISA-51 every 3 weeks for 8 courses,
among 9, 1 and 8 patients who completed the 8 vac-
be highly immunogenic. To identify those peptides
and intramuscular administration of poly-ICLC in
cinations spanning 24 weeks, 6, 1 and 3 patients in
we employ a dual approach.
HLA-A2 patients with: newly diagnosed high-risk
Cohorts 1, 2 and 3, respectively, are currently with
On the one hand MHC-ligands are isolated by
LGG without prior RT (Cohort 1); newly diagnosed
stable disease (with the median follow-up period
means of immunoaffi nity chromatography of MHC
high-risk LGG with prior RT (Cohort 2), or recur-
of 16.2 months).
molecules followed by acidic elution of peptides.
rent LGG (Cohort 3). Primary endpoints were safety
+
Conclusion: Our preliminary results demonstrate
These peptides are subsequently identified using
and CD8 T-cell responses against vaccine-targeted
that the regimen in these patients is well tolerated,
tandem mass spectrometry and database depend-
GAAs, assessed by Enzyme-Linked Immuno-SPOT
and induces robust type-1 anti-GAA T-cell respons-
ent search algorithms. On the other hand genomic
(ELISPOT) assays. Treatment response was evalu-
es. These data suggest that patients with LGG are
mutations forming the basis for tumor specific
ated clinically and by MR imaging. Targeted GAAs
suitable for vaccine therapy.
MHC-ligands are analyzed by next generation
+
were EphA2, interleukin (IL)-13 receptor-α2, sur204
1
Nico Trautwein , Markus Löffler ,Mathias Walzer , Derek Zieker , Philipp Horvath ,
3
2
2
2
1
Silvio Nadalin , Christopher Schroeder , Peter Bauer , Olaf Riess , Alfred Königsrainer ,
1
1
Hans-Georg Rammensee , Stefan Stevanović
2
exome sequencing. Based on sequencing results
205
152 | Therapeutic Vaccination
153 | Therapeutic Vaccination
Induction of antigen-specific T and B cell responses in mice
with a reconstituted human immune system
Vaccines for tumour prevention: Does Indoleamine 2,3-Dioxy­
genase (IDO) silencing enhance DNA vaccination?
1
1
3
2
1
1
3
3
2
Morad Zayoud , Khalifa El- Malki , Katrin Frauenknecht , Bettina Trinscheck , Helmut
2
3
1
1
Jonuleit , Clemens. Sommer , Ari Waisman and Florian C. Kurschus Marco Macagno , Elisabetta Bolli , Cristina Marchini , Augusto Amici , Chiara Riganti ,
2
1
1
Amalia Bosia , Guido Forni , and Federica Cavallo 1
Institute for Molecular Medicine
1
2
Department of Dermatology
Department of Clinical and Biological Sciences, Molecular Biotechnology Center, University of
Torino, Torino, Italy
3
Department of Neuropathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
2
Department of Genetics, Biology and Biochemistry Laboratory, University of Torino, Torino, Italy
3
Genetic Immunization Laboratory, Department of Molecular Cellular and Animal Biology,
­University of Camerino, Italy
Vaccination with antigen-pulsed dendritic cells
and IFN-γ when stimulated in vitro with myelin
As tumor progresses the efficacy of vaccination is
cellular and transmembrane domains of rat Erbb-2
(DCs) is a strategy, which has been employed to
antigens.
tuned down by suppressive activities. The admin-
(pVAX-ratECTM) and used for vaccination of BALB-
induce specific immune responses in trials to cure
In summary, we confirmed that the huPBL-NSG
istration of adjuvants or the silencing of specific
neuT mice carrying different stages of mammary
chronic infections as well as a treatment for cancer.
mice vaccinated with antigen pulsed DCs and then
immune regulatory molecules will optimize the
carcinogenesis.
The in vivo study of this system in current mouse
immunized with antigen in CFA can be used as
function antigen presenting cells (APC) and will
All the five interference cassettes were able to
models and the limitation of clinical studies in pa-
safe model to develop and study DC-based vaccina-
permit the immune response elicited to be active
reduce kynurenine release from N11 cells, con-
tients and their outcome gives only limited infor-
tion, and further to study the ability of the human
at the tumor site.
firming their ability to silence IDO expression.
mation on how to improve this therapy option to
immune response to these antigens and finally to
Indoleamine 2, 3-dioxygenase (IDO) the enzyme
Two cassettes were chosen to be subcloned into
finally obtain a benefit in the clinical treatment.
obtain a benefit for future clinical trials.
that degrades the essential amino acid tryptophan
pVAX-ratECTM, and used to vaccinate BALB-neuT
For bridging the gap between traditional mouse
in mammals is overexpressed in both tumor cells
mice bearing atypical hyperplasia and in situ car-
models and the human limited clinical trials we
and APCs in tumor-draining lymph nodes, where it
cinomas (weeks 10 and 12 of age) or microscopic
envisaged the development of a DC-based vaccina-
promotes the establishment of peripheral immune
invasive carcinomas (weeks 16 and 18). The in vivo
tion model in humanized mice, namely mice that
tolerance to tumor antigens. IDO seems to be an
observation of mammary cancer progression is still
contain a human immune system. For that purpose
ideal target to be silenced for the optimal induction
ongoing.
we humanized immunodeficient NOD/SCID/gc
206
-/-
of an antitumor immune response.
We expect that this simultaneous alteration of
(NSG) mice with human peripheral blood mononu-
We plan to use plasmids coding short shRNA spe-
tumor microenvironment and induction of an
clear cells (PBMCs), which permit the engraftment
cific for IDO to be administered together with the
immune response against Erbb-2 elicits an anti-
and expansion of human immune cells in vivo.
plasmid coding portion of Erbb-2, or plasmids con-
tumor response of therapeutic significance, in that
These mice were then immunized with a combina-
taining both the shRNA module and the oncoan-
it halts the progression of lesions that cannot be
tion of myelin-antigen pulsed DCs and active im-
tigen module, in vaccination-protection tests in
inhibited by Erbb-2 vaccination alone.
munization in Complete Freund’s Adjuvant (CFA)
BALB-neuT mice transgenic for the rat Erbb-2. Ret-
with the latter antigens.
roviral vectors (pLKO.1, Open Biosystem®) includ-
Starting at 18 days after the first DC-based priming,
ing five shRNA sequences targeting IDO mRNA
we found a specific weight loss in the antigen-im-
have been used as template to amplify the inter-
munized mouse group. We then found engraft-
ference cassettes (pU6-shRNA-IDO) that we cloned
ment of human B and T-cells in the spleen of these
into the Eco72I site of both pVAX1 (Invitrogen®).
mice, CNS cell infiltration as specific reaction to
The gene silencing efficacy of the various interfer-
myelin and human anti-myelin antibodies in the
ence cassettes was evaluated in a kynurenine assay
sera. Notably, T-cells recovered from these animals
using N11 microglial cells (Grant et al. 2000). The
showed specific proliferation and proinflammatory
most efficacious cassettes were subcloned into a
cytokine production such as IL-6, TNF-α, IL-17A
pVAX vector containing the sequence of the extra207
154 | Therapeutic Vaccination
155 | Therapeutic Vaccination
DCONE: Next generation off-the-shelf dendritic cell vaccine
­applicable for a broad range of cancer indications
Expansion of polyfunctional antigen-specific T-cells upon
­stimulation with mRNA electroporated dendritic cells in the
presence of immunomodulatory drugs
1
2
2
3
1
2
2
3
1
Sandra van Wetering , Saskia Santegoeds , Tanja de Gruijl , Gert Ossenkoppele ,
3
1
Arjan van den Loosdrecht , Ada Kruisbeek Brenda De Keersmaecker , Sabine D. Allard , Patrick Lacor , Rik Schots , Kris Thielemans 1
and Joeri L. Aerts 1
DCPrime BV, De Boelelaan 1085, 1081 HV, Amsterdam
1
Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium
2
Dept of of Medical Oncology
2
3
Dept of Haematology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam,
Netherlands.
Department of Internal Medicine and Infectious Diseases, Universitair Ziekenhuis Brussel,
Brussels, Belgium
3
Department of Clinical Hematology, Universitair Ziekenhuis Brussel, Brussels, Belgium
DCPrime is developing novel dendritic cell (DC)
In addition, upon pulsing with antigenic peptides
Since its FDA approval, thalidomide/dexametha-
ciated with a better HIV disease control. Further-
vaccines based on its proprietary DCOne cell line.
of choice, mature DCOne cells efficiently present
sone combination therapy has been successfully
more, CD8 T-cells responded to lower antigenic
This DCOne is a precursor cell for dendritic cells,
them to T-cells, and can do so equally well after
used in first-line multiple myeloma treatment.
peptide concentrations and recognized a higher
and is used to generate ‘off-the-shelf’, as opposed to
freezing and thawing.
More active and less toxic thalidomide-derivatives
number of Gag epitopes upon addition of IMiDs.
patient-based, dendritic cell based vaccine. DCOne
DCPrime’s lead product DCP-001, which consists
such as lenalidomide and pomalidomide have been
As opposed to the CD8 T-cell proliferation, IMiDs
originates from a human myeloid leukemia precur-
of matured DCOne cells, is applicable for various
developed. Thalidomide and its derivatives exert
reduced the proliferation of the HIV-specific CD4
sor cell line and endogenously expresses multiple
tumour indications but is currently being tested
direct anti-tumor effects but are also strong stimu-
T-cells while increasing the number of polyfunc-
tumour associated antigens.
in an open label dose-escalation Phase I/IIa study
lators of NK-cell and polyclonal T-cell responses
tional CD4 T-cells, be it to a lesser extent com-
DCPrime has developed reproducible procedures to
in Acute Myeloid Leukemia (AML) patients. This
and are therefore referred to as immunomodula-
pared to the CD8 T-cells.
make fully functional DC out of DCOne, such that
study will provide essential safety and immuno-
tory drugs (IMiDs). We hypothesize that the immu-
These results provide new information about the
it can become a more patient friendly alternative
logical monitoring data, in preparation for subse-
nostimulatory properties of IMiDs could improve
effects of IMiDs on antigen-specific T-cells and
to the current cumbersome patient-based DC-based
quent Phase II and III studies in AML. Currently, 6
the efficacy of immunotherapies, such as dendritic
suggest that these drugs might increase the effi-
products. Potential functionality of the DCOne cell
patients have completed four cycles of vaccination
cell-based vaccines. In this study, we therefore aim
cacy of immune therapies for infectious diseases
line to serve as the basis for dendritic cell based
and treatment was well tolerated with no product
to investigate the effects of IMiDs on antigen-spe-
and cancer.
cancer vaccines, has been established in multiple
related adverse events, indicating that treatment is
cific T-cell responses in vitro.
in vitro assays. Essentially, matured DCOne cells
feasible and safe. Systemic immune responses such
Since it has been demonstrated that disease pro-
exhibit all the properties of functional mature DCs
as Delayed Type Hypersensitivity reactions (DTH),
gression during HIV infection is not merely deter-
derived from blood borne precursors and display
T-cell infiltrates (CD4 and CD8 T-cells) within the
mined by the number of HIV-specific T-cells, but
a specific repertoire of DC surface molecules that
DTH sites, and antibody responses have been noted
rather by their quality, strategies to specifically
are widely used and accepted as the DC hallmarks
in several patients. Additional immune biomarker
enhance or induce high quality HIV-specific T-cell
associated with mature and fully functional den-
and clinical response data will be collected in the
responses are necessary to develop effective HIV
dritic cells. Pertinent functional characteristics
next few months.
immune therapies. Therefore, we decided to evalu-
are also expressed by mature DCOne cells: They
ate the effects of lenalidomide and pomalidomide
display MHC-class I and II antigens essential for
on HIV-specific T-cells. We found that the presence
antigen processing and presentation, they migrate
of IMiDs during in vitro T-cell stimulation with den-
in response to lymph node homing chemokines,
dritic cells electroporated with Gag or Nef encoding
induce T-cell proliferation, induce specific cytokine
mRNA resulted in an increased HIV-specific CD8
T-cell proliferation and cytokine (IFN-γ, TNF-α and
response towards a Th1/Th17 response, and can
IL-2) production. Interestingly, the IMiDs particu-
+
T-cells that
are subsequently able to kill tumour targeT-cells.
208
+
+
+
+
+
release by T-cells indicating skewing of the T-cell
prime tumour antigen-reactive CD8
+
+
larly enhanced polyfunctional HIV-specific CD8
T-cells, which were previously shown to be asso209
156 | Therapeutic Vaccination
157 | Therapeutic Vaccination
Targeting Cancer Stem Cells by raising cytotoxic T-cell
­responses against the stemness protein SOX2
Comprehensive preclinical model evaluating a protein-based
PRAME specific cancer immunotherapy to fight against PRAME
expressing tumors
Lukasz Bialkowski, Brenda De Keersmaecker, Sandra Van Lint, Sarah Maenhout, ­
Kevin Van der Jeught, Carlo Heirman, Kris Thielemans, Joeri Aerts
Catherine Gérard, Nathalie Baudson, Thierry Ory, Romain Piccininno, Aurelie Delplanque,
Carole François, Lawrence Segal, Jamila Louahed
Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Laarbeeklaan 103E,
1090 Brussels, Belgium
GlaxoSmithKline Biologicals, Rixensart, Belgium
210
Despite significant advances one of the major chal-
By means of in vivo cytotoxic T lymphocyte (CTL)
Background: The human tumor antigen PRAME
specifically protected against a tumor challenge
lenges in oncology remains the frequent recurrence
assays, we show that mice immunized with SOX2-
(PReferentially expressed Antigen of MElanoma) is
with PRAME-expressing tumor cells with a long
of tumor, even in the absence of clinical signs of
TriMix mRNA developed SOX2-specific CTL re-
expressed in a variety of cancers. This antigen rep-
lasting memory when the challenge was applied
neoplasia. Growing evidence indicates that cancer
sponses. Additionally, using overlapping 15-mer
resents an excellent target for immunotherapy as its
long after the last immunization (2 months).
relapse is likely driven by a rare population of cells
peptides (n=86), we identified regions within the
expression is shared by different tumor types. The
Experiments in HLA-A02.01/HLA-DR1 transgenic
endowed with stem cell characteristics lurking
SOX2 protein that are the most immunogenic.
development of a PRAME cancer immunotherapy
mice showed the induction of both anti-PRAME
within treated primary tumor beds and initiating
Preliminary ELISA and intracellular cytokine stain-
able to induce strong T-cell responses would be a
CD4 and CD8 T-cell responses.
tumors at distant places. Therefore, specific target-
ing (ICS) experiments performed on spleno-cytes
specific targeted therapy and could provide signifi-
A repeated dose toxicity study conducted with non-
ing of these cancer stem cells (CSCs) could turn
isolated from immunized mice, showed that in re-
cant benefits to a large number of cancer patients.
human primates has shown that the PRAME Im-
a short-term remission into long-term disease-free
sponse to in vitro restimulation these cells secrete
Methods: In these studies we used a mouse tumor
munotherapy was well tolerated and did not induce
survival for many cancer patients.
cytokines such as IFN-γ and IL-2 and exhibit a lytic
genetically modified to express PRAME. We char-
any signs of systemic toxicity.
One of the major character-ristics of CSCs is their
capacity in vitro against targeT-cells expressing the
acterized the immune response and anti-tumor
Conclusion: Preclinical results showed that PRAME
expression of stemness genes, such as SOX2, Nanog
antigen of interest.
activity following repeated injections of a PRAME
immunotherapy consistently induced a compre-
and OCT4. It has been shown that these transcrip-
We are currently trying to identify specific epitopes
recombinant protein formulated with an im-
hensive immune response and provided protection
tion factors play an important role in maintaining
within the SOX2 sequence. This should facilitate
munostimulant. We also evaluated the immune
of mice against tumor challenge. This supports the
the stemness of embryonic stem cells and become
future experiments, as defined peptides can be
response induced by PRAME in HLA-A02.01/
choice to use an immunostimulant in combination
reactivated in several human cancers. Moreover,
used for immunization and read-out, e.g. by gener-
HLA-DR1 transgenic mice. The safety and tolerabil-
with the PRAME protein for future clinical develop-
monoclonal gammopathy of undetermined signifi-
ating SOX2-specific tetramers.
ity of the PRAME immunotherapy was assessed by
ment. The PRAME immunotherapy has now moved
cance (MGUS) patients display significant levels of
In summary, we have shown that SOX2-specific
repeated injections in cynomolgus monkeys.
into Phase I development in melanoma and NSCLC.
T-cell immunity against the stemness protein SOX2.
CTLs can be raised in mice by intranodal mRNA
Results: The experiments conducted in mice dem-
Therefore we decided to investigate the potential of
injection. We will now pinpoint the epitopes and
onstrated that the PRAME protein was weakly
SOX2 as a target for anti-cancer vaccination.
evaluate whether these CTLs are capable of recog-
immunogenic by itself and that the addition of
In a first instance we evaluated the immunogenic-
nizing CSCs in vitro. Ultimately, we want to treat
an immunostimulant was required to induce a
ity of SOX2 in mice. To that aim we employed a
mice inoculated with CSCs by targeting SOX2.
comprehensive immune response. This response
novel immunization technique in which mRNA
included 1) the generation of PRAME-specific anti-
encoding a given antigenic peptide is directly in-
bodies with a Th1 isotypic profile, and 2) the induc-
jected into inguinal lymph nodes, along with a
tion of PRAME-specific CD4 T-cells that were able
mixture of mRNAs encoding CD40L, constitutively
to produce cytokines IFNγ, TNFα) in response to the
active TLR4, and CD70 (TriMix). DCs matured with
antigen. The immune response induced was sys-
TriMix mRNA were shown to be capable of effi-
temic as it could be identified in lymphoid organs
ciently stimulating antigen-specific T-cells.
and in the blood. Moreover, immunized mice were
+
+
+
211
158 | Therapeutic Vaccination
159 | Therapeutic Vaccination
Combining common chemotherapeutic regimens with immunotherapy – assessment of the immunological effects of FOLFOX,
FOLFIRI and cisplatin
RNAdjuvant®: a novel, highly-potent, RNA-based adjuvant
­supports induction of balanced immune response (TH1 and
TH2) and anti-tumor activity
Nina Pawlowski, Helen Hörzer, Toni Weinschenk, Harpreet Singh, Norbert Hilf
Regina Heidenreich, Mariola Fotin-Mleczek, Patrick Baumhof, Birgit Scheel, Söhnke Voss,
Thomas Kramps, Karl-Josef Kallen
immatics biotechnologies GmbH, Tübingen, Germany
CureVac GmbH, Tübingen, Germany
212
In the past the predominant opinion was that
or concurrent with each vaccination. Two weeks
For efficient cancer immunotherapy, strong adju-
ing the shift towards TH1 response as determined
chemotherapies (CTs) are immunosuppressive and
after the first immunization, induction of OVA-
vants are mandatory to induce a potent and per-
by increased titers of IgG2a antibodies.
therefore a combination of chemotherapy with
001-specific T-cell was analyzed flow cytometry
sistent immune response, particularly as tumor
Peptide-based vaccines, including Ovalbumin-de-
immunotherapy would be not advisable. Today,
after MHC multimer staining as well as by IFN-
associated antigens used as purified proteins or
rived SIINFEKL epitope and Human Papillomavi-
this dogma has been questioned by recent find-
gamma ELISPOT.
peptides show mostly only a weak intrinsic im-
rus (HPV)-derived peptides, also strongly benefit-
ings showing that several CTs can be used to even
No significant (negative or positive) effect of OX- or
munogenicity. CureVac have recently developed
ed from RNAdjuvant® and exhibited significantly
modulate the immune system and/or the tumor
CDDP-based treatment on CTL induction if chemo-
a novel RNA-based adjuvant with strong immu-
higher T-cell responses compared to non-adjuvant-
microenvironment in a favorable way, thereby fa-
therapy was applied 3 days before vaccination. If
nostimulatory properties. RNAdjuvant® is physi-
ed counterparts.
cilitating the control of tumor progression by the
chemotherapy and vaccination were applied simul-
cally and chemically well-defined and exhibits a
Tumor challenge experiments revealed a complete
patient’s immune system or by immunotherapies
taneous, there was a significant decrease of CTL
very good safety profile. RNAdjuvant® is well tol-
protection of mice vaccinated with RNAdjuvant®
as for example cancer vaccines. Biweekly applied
induction in mice treated with CDDP-based, but
erated even at high doses and does not induce in
in combination with either recombinant protein
oxaliplatin (OX)- and irinotecan (IR)-based CTs
not with OX- or IR-based CT. Moreover, treatment
mice the splenomegaly described for standard ad-
(Ovalbumin) or peptides (HPV-derived), whereas
(e.g. FOLFOX or FOLFIRI) are standard treatments
of mice with CDDP 3 days before vaccination com-
juvants such as CpG-DNA. RNAdjuvant® supports
vaccination with protein or peptides alone had no
for colorectal cancer while cisplatin (CDDP)-based
bined with repeated daily 5-FU application (day -3 to
both antigenic formats of recombinant proteins and
impact on tumor growth. Importantly, vaccination
regimens are common in the treatment of gastric
day +1) significantly inhibited immune responses.
peptides, causing dramatically increased immuno-
with RNAdjuvant® in combination with both anti-
cancers, but only few reports have examined the
Although OX- or IR-based CTs had no significant
genicity of these vaccines, especially in respect of
genic formats led to a significant growth inhibition
effects of these CT regimens on the immune system.
inhibitory influence on vaccine-induced immune
T-cell responses and anti-tumoral activity. In vitro
of already established tumors.
Here, we evaluated the influence of common CT
responses in our mouse experiments, additional in
studies in human peripheral blood monocytes dem-
Taken together our data demonstrate that RNAdju-
compounds (OX, IR, 5-fluorouracil (5-FU) plus leu-
vitro analyses (mixed lymphocyte reaction) using
onstrated that RNAdjuvant® is preferentially taken
vant® represents a novel breakthrough technology
coverin (LV) or CDDP) on vaccine-induced immune
human PBMCs showed an inhibitory effect of OR
up by antigen presenting cells (APC) resulting in
which may revolutionize the field of cancer vac-
responses in vivo. C57BL/6 mice were immunized
and IR on CD4 and CD8 T-cell proliferation when
a strong APC activation. RNAdjuvant® induced
cines requiring safe and potent adjuvants.
subcutaneously (s.c.) twice (day 0 and day 7) with
the cytotoxic agents were used in clinically rele-
activation of APC led to an increased expression
50 µg of SIINFEKL (OVA-001) peptide alone (nega-
vant concentrations.
of specific activation markers and secretion of cy-
tive control) or combined with 200 µg Poly-IC as
In summary, these results suggest that immuno-
tokines driving a pronounced TH1 response. In ad-
adjuvants (positive control). Some mice received
therapy can be combined with chemotherapy regi-
dition, RNAdjuvant® stimulates the innate immune
additionally two intraperitoneal (i.p.) applications
mens without reducing immunological efficacy,
response by activation of (natural killer) NK cells
of OX-based (10 mg/kg 5-FU + 5 mg/kg LV + 5
but that the timing of applications has to be care-
and IL-12 secretion.
mg/kg OX per cycle, resembling FOLFOX regimen
fully chosen. Due to the anti-proliferative effects
In vivo, vaccination with RNAdjuvant® in combi-
in humans), IR-based (10mg/kg 5-FU + 5 mg/kg
in vitro and the slight decrease in CTL induction
nation with recombinant protein, e.g. Ovalbumin,
LV + 20 mg/kg IR per cycle, resembling FOLFIRI)
by concurrent application in vivo, patients should
elicited strong antigen-specific cytotoxic T-cell re-
or CDDP/5-FU based (1 or 5 daily applications of
be immunized at the earliest 48h after CT to avoid
sponses, which are barely induced by vaccination
10mg/kg 5-FU + application of 5 mg/kg CDDP
negative effects of CT on vaccine-induced immune
with recombinant protein alone. RNAdjuvant® also
per cycle) CT regimens 3 days before vaccination
responses.
enhanced the humoral response strongly support213
160 | Therapeutic Vaccination
161 | Therapeutic Vaccination
Dendritic cell vaccination in melanoma patients: mRNA
­electroporated dendritic cells improves immunological and
­clinical responses
IVAC - Individualized Vaccines for Cancer
1, 2
1, 2
1
2
1
Kalijn F. Bol , Erik H. Aarntzen , Gerty Schreibelt , W. Joost Lesterhuis , Carl G. Figdor ,
2
1
1
Cornelis J. Punt , Jolanda M. de Vries , Gosse Adema 1
2
Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Nijmegen,
Netherlands.
Department of Medical Oncology, Radboud University Nijmegen Medical Centre, Nijmegen,
Netherlands.
1
1
1
1, 2
Niels van de Roemer , Jan Diekmann , Sebastian Kreiter , Abderrouf Selmi ,
1
1, 2
1, 2
1
1, 2
Mustafa Diken , Marc Holzmann , René Roth , Cedrik Britten and Ugur Sahin
1
Center for Translational Oncology and Immunology (TRON)
2
Experimental & Translational Oncology, Department of Internal Medicine III, Group of
Prof. U. Sahin / PD Dr. Ö. Türeci, Johannes Gutenberg University Mainz, Germany
Immunotherapy exploiting ex vivo generated au-
Cancer is driven by multiple genetic events followed
mutations by whole exome sequencing. After se-
tologous dendritic cells (DC) has proven proof of
by further clonal evolution, making disease elimi-
lection of high expressed genes and potential
principal in clinical trials. We, and others, have
nation with single targeted drugs a difficult task.
MHC binding affinities of the respective mutated
shown that tumor-specific immune responses can
The multiplicity of gene mutations derived from
epitopes 50 mutations were chosen and validated
be induced in stage III and IV melanoma patients.
sub-clone heterogeneity may represent an ideal
by Sanger-Sequencing. For these 50 mutations, we
The majority of clinical studies in melanoma pa-
setting for multi-epitope tumor vaccination. Vac-
wanted to define the immunogenicity of the muta-
tients have been performed with MHC class I re-
cination is a powerful method to induce humoral
tion coding sequences. To this end, we designed
stricted peptide-pulsed DC. There are at least theo-
and cellular adaptive immune responses aiming
27-meric peptides incorporating either the mutated
retical disadvantages of this method. Firstly, the
to control bacterial and viral infections as well as
or the wildtype amino acid centrally and immu-
use of MHC class I restricted peptide epitopes and
tumor growth. Whereas the prevention of infections
nized C57BL/6 mice with the mutated peptides.
+
cytotoxic T-cells only,
relies on neutralizing antibodies, the treatment of
Using IFNγ ELISpot we found one third (16/50) of
without involving CD4 T helper cells in the induc-
chronic intracellular infections and cancer strongly
them were shown to be immunogenic. Of these,
tion of specific anti-tumor responses. Furthermore,
+
depends on the activation of antigen-specific CD4
60% elicited immune responses preferentially di-
the peptide epitopes may dissociate from MHC
and CD8 T-cells. Vaccines are particularly suited
rected against the mutated sequence as compared
molecules due to low affinity and MHC turnover
for precise targeting of tumor associated molecular
to the wild type sequence. In the next step, we
and it does not account for post-transcriptional
alterations. In the last years, multiple tumor- asso-
tested the anti tumor potency of the new B16-F10
modifications of peptide epitopes. A strategy to
ciated antigens (TAA) have been identified. Several
epitopes in a tumor transplant model. Immuniza-
circumvent most of the disadvantages of peptide
categories of TAA have been described based on
tion with peptides conferred in vivo tumor control
pulsing, is mRNA electroporation. In our clinical
tumor-associated deregulation of gene expression.
in protective and therapeutic settings, qualifying
trial we investigated the immunological and clini-
Furthermore, neo-epitopes can be created by non-
mutated epitopes containing single amino acid sub-
cal responses to intranodal vaccination with DC
synonymous, somatic mutations. As they are not
stitutions as effective vaccines. Our study provides
electroporated with mRNA encoding for gp100 and
subject to central immune tolerance, these muta-
a comprehensive picture of the B16-F10 mutanome
tyrosinase. We show that electroporated DC retain
tions can be ideal candidates for individual vaccine
and gives a first insight into the immunogenicity
their phenotype and maturation potential and that
development. Human cancers carry 100-300 non-
of non-synonymous point mutations. The combina-
they are able to induce functional, IFNγ producing,
synonymous mutations on average. Our starting
tion of deep sequencing with systematic analysis
hypothesis is that cancer can be targeted by T-cells
of the immunogenicity paves a new path for the
induced by poly-neo-epitopic vaccines based on
individualized immunotherapy of cancer patients.
therereby targeting CD8
+
+
tumor-specific CD8 cytotoxic T-cells in a subset of
+
our patients as well as CD4 tumor-specific cells.
+
non-synonymous individual tumor-specific mutations. For testing this hypothesis, we resorted to
B16-F10 murine melanoma for which our team
has identified more than 500 non-synonymous
214
215
162 | Therapeutic Vaccination
163 | Therapeutic Vaccination
Intradermal immunization with a novel mRNA-based vaccination technology induces strong T- and B-cell responses in phase
I/IIa trials in non-small-cell lung cancer (NSCLC) and prostate
carcinoma (PCA)
Immunological results of a phase 1-2 therapeutic ­vaccination
study by MGN1601 in patients with advanced renal cell
­carcinoma
1
2
3
4
5
Martin Sebastian , Hubert Kübler , Arnulf Stenzl , Kurt Miller , Alfred Zippelius , Wolfgang
6
3
7
8
9
Schultze-Seemann , Frank Mayer , Martin Reck , Djordje Atanackovic , Frank vom Dorp , Lotta
11
10
10
10
11
von Boehmer , Birgit Scheel , Sven D. Koch , Thomas Lander , Alexander Knuth , Karl-Josef
10
Kallen 1
Med. Klinik,Universität Mainz Mainz, Germany,
2
Klinikum rechts der Isar der TU München, München, Germany
3
Universitätsklinik Tübingen, Tübingen, Germany
4
Charité - Universitätsmedizin Berlin, Berlin, Germany
5
Universitätsspital Basel, Basel, Switzerland
6
Universitätsklinikum Freiburg, Freiburg, Germany
7
Krankenhaus Großhansdorf, Hamburg, Pneumologie und Thoraxchirurgie, Germany
6
Universitätsklinik Hamburg-Eppendorf, Hamburg, Germany
9
Universitätsklinikum Essen, Essen, Germany
10
CureVac GmbH, Tübingen, Germany
11
UniversitätsSpital Zürich, Zürich, Switzerland
1
2
3
1
Mologen AG, Berlin, Germany
2
Charité University Medicine, Clinic for Urology, Department for Internal Medicine, Berlin, Germany
3
Medical University Hannover, Department Hematology and Oncology, Germany
4
University of Bonn, Department of Medicine III, Center for Integrated Oncology, Bonn, Germany
5
Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Germany
Background: In this first-in-man phase 1-2 clinical
Results: Of nineteen patients with heavily pre-
trial (ASET study) safety, efficacy, and immuno-
treated advanced stage RCC, ten completed the
genicity of the cell-based therapeutic cancer vaccine
treatment phase per protocol (TPP). Four out of 19
MGN1601 were evaluated. MGN1601 consists of two
intended to treat patients (21%) achieved disease
CV9201 and CV9103 are novel cancer vaccines against
quency was observed. A confirmed PSA decline > 80%
active pharmaceutical ingredients in fixed combi-
control (one PR and three SD) after 12 weeks ac-
NSCLC and castrate-resistant PCa composed of self-
was observed in 1 patient.
nation: fourfold gene-modified, allogeneic tumor
cording to RECIST 1.1 and irRC criteria. In the
adjuvanting mRNA molecules that are administered
CV9201 was tested in NSCLC patients (stage IIIb/
cells expressing IL-7, GM-CSF, CD80, and CD154
TPP population the rate of patients with disease
intra-dermally. Both trials were open-label, uncon-
IV) with at least stable disease after 1st line chemo-
through MIDGE® gene expression vectors and the
control was 40% (4 out of 10). In these patients,
trolled, multi-center, international, prospective, in-pa-
therapy or chemoradiotherapy. Treatment related AEs
DNA-based immunomodulator dSLIM® as a TLR-9
the frequencies of tumor-specific IFN-γ secreting
tient phase I/II studies with the primary objective in
were mainly grade 1/2 injection site reactions or fever.
agonist. Patients with advanced renal cell carcino-
CTL and the titres of vaccine-specific antibodies
phase I being dose finding for the phase II part, assess-
Neither dose-limiting toxicities nor related serious AEs
ma (RCC), who failed standard therapy lines, were
did increase with increasing rounds of vaccina-
ment of safety of the trial regimen and evaluation of
were observed. 31 patients received up to 5 vaccina-
included in this study.
tion. The frequencies of monocytic MDSC in PMBC
induction of immune response. In Phase II, the respec-
tions and were evaluated for the induction of immune
Methods: The ASET study was designed as a mul-
slightly decreased during vaccination in those pa-
tive objective was assessment of safety, evaluation of
responses two weeks after the 3 and 5 vaccination.
ticentric, open, single-arm phase 1-2 clinical trial.
tients, who achieved disease control, whereas this
induction of immune response and assessment of anti-
Ag-specific B- or T-cell responses were assessed using
During the treatment phase the study drug was
cell population increased in patients with progres-
tumor activity. CV9201 is composed of mRNAs coding
the before mentioned methods. Ag-specific immune re-
administered eight times; the first three applica-
sive disease (PD) after 12 weeks of treatment. The
for NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4;
sponses were detected in 65% of patients, 56% thereof
tions in weekly intervals, and the following five ap-
CD3+ T-cell frequencies increased or were stable
CV9103 of mRNAs coding for PSA, PSCA, PSMA and
were multiple responders. Remarkably, a significant 2.5
plications every two weeks. Patients who achieved
in the patients with disease control, but decreased
STEAP-1. Blood samples were taken before and vac-
- 13 fold shift from naïve B-cells to pre-germinal center
disease control (defined as CR, PR or SD) were pro-
in patients with PD following the course treatment.
cination. Immune response was assessed by ELISPOT
B-cells was detected in 61% of patients. The presence of
posed for an extension phase to receive 5 further
Conclusions:
(IFN-γ), intracellular cytokine staining (IFN-γ, TNFα,
ag-specific B cells was demonstrated exemplarily with
applications up to week 120. During treatment, the
MGN1601 shows promising efficacy in late stage
rd
th
The therapeutic cancer vaccine
IL-2), tetramer analysis (ex vivo) or ELISA.
a B-cell proliferation assay. Altogether, 84% of patients
following immunological parameters were evalu-
RCC patients, and the pronounced radiological
The final data analysis of CV9103 vaccination in PCa
showed an immune reaction after vaccination.
ated: frequency of cytotoxic T-cells (CTL), as de-
response corresponds well with the measured
patients revealed favorable safety with possibly related
These results strongly suggest that intradermal immu-
tected by ELISPOT, frequencies of myeloid-derived
immune reactions. Further clinical studies with
serious adverse events (AE) observed in only 2 out of
nization with self-adjuvanting mRNA vaccines consti-
suppressor cells (MDSC), regulatory T-cells (Treg)
MGN1601 are on their way.
44 patients. Immune monitoring demonstrated antigen
tutes a safe, flexible and highly immunogenic, novel
and other immune cell populations analysed by
(ag)-specific T- and/or B-cell responses against at least
vaccination approach able to induce antigen-specific
flow cytometry. Additionally cytokines and anti-
one antigen in 79% of the evaluable patients. Impor-
humoral and cellular immune responses in the major-
tumor cell antibodies were analysed. Radiological
tantly, 63% of responders reacted against more than
ity of PCa as well as NSCLC patients.
efficacy data were evaluated according to RECIST
one antigen. Anon-significant increase of B-cell fre216
1
Kerstin Kapp , Ekaterina Weith , Steffen Weikert , Viktor Gruenwald , Ingo G. H. Schmidt4
1
1
1
5
Wolf , Manuel Schmidt , Matthias Schroff , Marina Tschaika , Burghardt Wittig 1.1 and immune related Response Criteria (irRC).
217
164 | Therapeutic Vaccination
165 | Therapeutic Vaccination
Long Peptides Complexed with A Novel Delivery Sytem CHP
Nanogel Leads to the Improved Vaccine-induced Specific
­Immune Responses with CpG oligo DNA or poly-I:C RNA
Preliminary results of a triple peptide escalating dose
­vaccination Phase I/II clinical trial as consolidation treatment
in women affected by ovarian cancer
1, 3
1, 3
1, 3
1
4
1
Naozumi Harada , Daisuke Muraoka , Tae Hayashi , Koji Yoshimi , Shin-ichi Sawada ,
4
1, 2
Kazunari Akiyoshi and Hiroshi Shiku 1
Department of Cancer Vaccine and Mie University Graduate School of Medicine, Mie, Japan
2
Departments of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie,
Japan
3
Basic and Preclinical Research Department, ImmunoFrontier, Inc., Tokyo, Japan
4
Department of Polymer Chemistry, Kyoto University Graduate School of Technology, Kyoto,
Japan
Targeted delivery of vaccine antigens such as pep-
2
1
CHP nanogel, the long peptides were promptly in-
1
Department of Experimental Medicine, University Sapienza, Rome, Italy
2
Department of Gynaecology, Obstetrics and Urology, University Sapienza, Rome, Italy
Ovarian cancer remains one of the most lethal
1
at 3 months from the sixth dose. Results revealed
tides or proteins to lymph nodes could be an effec-
corporated to the significant proportion (approx. 10
malignancies in developed countries and recent-
the vaccine to be safe and feasible in ovarian cancer
tive strategy to potentiate the immunogenicity and
to 55%) of CD11c dendritic cells or CD11b mac-
ly no improvement in disease free survival (DFS)
patients. Side effects accounts for ECOG scale 1-2
efficacy of therapeutic cancer vaccines. Synthetic
rophages in the DLN, while the long peptides with
and overall survival (OS) has been numbered for
levels signs (itch, erythema and tumescence at
IFA were not. Detailed analysis of the CHP nanogel
conventional therapies . Immunotherapy could be
injection site) and symptoms (fever, fatigue and
+
long peptides (~40 aa) containing both CD8 T-cell
+
+
+
2
T-cell epitopes derived from human
mediated-transportation of long peptides to the
employed as consolidation treatment after standard
malaise), for both the dose-administered. Good
MAGE-A4 or murine mutated ERK2 antigens were
DLN suggested that the transportation involved a
therapies in patients in which disease and immune
compliance was found to vaccine schedule in all
complexed with a nanogel formed by self-aggrega-
cell-independent mechanism in part.
suppression is minimal. Furthermore, epitope
patients enrolled. Secondary endpoints results re-
tion of cholesteryl hydrophobized pullulan (CHP).
These results indicate that an antigen delivery
spread and new adjutants have been found to sig-
vealed a 3 years 50% (3/6 patients) DFS and a 83%
Either the long peptide/CHP nanogel complex or
system of CHP nanogel, termed “immunotrans-
nificantly increase the efficacy of novel immuno-
(5/6 patients) Os in FIGO stages IIIc-IV ovarian
the long peptide admixed with incomplete Fruend’s
porter” by efficiently transporting vaccine peptides
therapic strategies.
cancer patients (60%; 6/10 patients). Moreover,
adjuvant (IFA) was injected subcutaneously to
to the DLN, is critical for effective cancer vaccines.
This Phase I/II study aimed to improve vaccine
immunological and clinical correlation analysis re-
potency and enhance immune response of two
vealed a significant increased IFNγ CD8 specific
CD8 T-cells and CD4 T-cells was comparatively
mucins (MUC-1 and CEA) and Erb-B2 tumour asso-
T-cells production along with vaccination steps
measured. Induction of both specific T-cells was
ciated antigens (TAAs), with the co-administration
(χ =6.67; p<0.009) in the subgroup of patients who
at marginal levels in two groups. However, when
of a new adjuvant combination: Montanide ISA51
did not recur vs. controls (mean follow up: 845.1
both vaccine groups were co-administered with
plus GM-CSF. Keyhole Limpet Hemocyanin (KLH)
days; range 55-1523).
Toll-like receptor (TLR) agonist such as CpG oligo
was adopted as immunological tracker.
The highest number of vaccine induced specific
DNA or poly-I:C RNA, T-cell activities became more
Ten disease-free HLA-A2 aplotype high-risk serous
CD8 cells were found at the end of the 6 doses
prominent only in those with the long peptide/CHP
advanced stage ovarian cancer patients underwent
although high levels of T-cells could be also re-
nanogel. The inhibitory effect of vaccination on the
a two-groups safety clinical trial vaccination pro-
induced by the recall boost. A significantly higher
in vivo growth of tumors expressing targeted anti-
tocol at “Sapienza” University of Rome from 2007
specific immune response was observed in the
gens was also greater in the mice immunized with
to 2010. CEA, Erb-B2 and MUC1 expression rates
group B.
the long peptide/CHP nanogel than those with the
on primary tumours were 40%, 30% and 100%, re-
long peptide/IFA.
spectively. Group A (8 patients) received 100μg of
1. Jemal A. CA Cancer J Clin. 2009;59:225.
Capability of CHP nanogel to transport subcutane-
each peptide per dose accordingly to the immuno-
2. Pecorelli S. J Clin Oncol. 2009;27:4642.
ously injected long peptides to the draining lymph
histochemically antigen expression, whereas Group
node (DLN) was evaluated. Incorporation of the
B (2 patients) was vaccinated with all the peptide
fluorescently labeled long peptides into the anti-
regardless TAAs expression at the dose of 500μg
gen-presenting cells in the DLN was analyzed by
each. Vaccination schedule included 6 consecutive
flow cytometry. As a result, when complexed with
peptide vaccine administrations and a recall dose
and CD4
BALB/c mice, and the induction of peptide-specific
+
218
1
Valeria Visconti , Hassan Rahimi Koshkaki , Morena Antonilli , Chiara Napoletano ,
2
1
2
1
1
Milena Pernice , Giacomo Barchiesi , Ilary Ruscito , Ilaria Grazia Zizzari , Aurelia Rughetti ,
2
2
1
Filippo Bellati , Pierluigi Benedetti Panici , Marianna Nuti .
+
+
2
+
219
166 | Therapeutic Vaccination
167 | Therapeutic Vaccination
Immune responses against frameshift antigens in microsatellite
unstable colorectal cancers
Sipuleucel-T product characterization across different disease
states of prostate cancer
1
1
1
2
1
2
3
1
1
Miriam Reuschenbach , Matthias Kloor , Kathrin Bauer , Reza Rafiyan , Salah-Eddin Al
2
2
2
1
Batran , Julia Karbach , Elke Jäger , Magnus von Knebel Doeberitz Johnna Wesley , David Quinn , Celestia Higano , Heather Haynes , Frances Stewart ,
1
1
1
­Christian Poehlein , James Trager , Nadeem Sheikh 1
Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg
1
Dendreon, Seattle, WA 98101, USA
Hämatologie-Onkologie, Krankenhaus Nordwest, Frankfurt, Germany
2
Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA
90089, USA
3
School of Medicine, University of Washington, Seattle, WA 98105, USA
2
220
Colorectal cancer is a heterogeneous tumor type.
established as potent tumor antigens. Moreover, we
Background: Sipuleucel-T is an autologous cellu-
ic or minimally symptomatic mCRPC: 28.7, mCRPC:
Whereas the majority of colorectal cancers show
could show that FSPs are capable of inducing spon-
lar immunotherapy approved for the treatment of
21.8; p<0.0001 (Joncheere-Terpstra test)). During
chromosomal instability, a subset of about 15% of
taneous cellular and humoral immune responses
asymptomatic or minimally symptomatic metastat-
manufacture of the product, lymphocyte activa-
colorectal cancers (CRC) have a deficient DNA mis-
in patients with MSI-H CRC. Interestingly, cellular
ic castrate resistant prostate cancer (mCRPC). In
tion markers and cytokines consistently showed
match repair (MMR) system and accumulate small
and humoral immune responses against FSPs can
the Phase 3 mCRPC trials, antigen presenting cell
enhanced expression during the second and third
mutations at repetitive DNA sequences, a pheno-
be detected not only in patients with MSI-H CRC,
(APC) activation in sipuleucel-T correlated with
treatments in all disease states. The lymphocyte ac-
type termed high level microsatellite instability
but also in healthy Lynch syndrome mutation car-
overall survival. Here product characteristics of
tivation and cytokine profiles were similar between
(MSI-H). MSI-H cancers are particularly charac-
riers without clinically manifest tumors.
sipuleucel-T were compared in patients (pts) from
early and later disease state pts.
teristic for individuals with the inherited HNPCC
MMR deficiency-induced FSP antigens therefore
trials in the neoadjuvant (n=42), asymptomatic
Conclusions: While cellular composition varied
(hereditary non-polyposis colorectal cancer) or
represent promising target structures for therapeu-
or minimally symptomatic mCRPC (n=341), and
with disease state, manufacture of sipuleucel-T ac-
Lynch syndrome, which is caused by germline mu-
tic vaccination of MSI-H CRC patients.
mCRPC (n=104) disease states.
tivated APCs in both early and late disease states of
tations of the MMR genes. Mutation carriers have
Recently a phase I/IIa peptide vaccination trial has
Methods: Sipuleucel-T comprises 3 treatments ap-
prostate cancer, and generated similar T and B cell
a high lifetime risk for the development of MSI-H
been initiated to evaluate the safety and immuno-
proximately 2 wks apart. Cellular composition and
activation and cytokine production profiles consist-
cancers. Dense infiltration with lymphocytes is
genicity of three FSP antigens upon administration
APC activation (CD54 upregulation) were evalu-
ent with immunological prime-boost. APC activa-
commonly observed in MSI-H CRC lesions. The
to patients with UICC stage III or IV MSI-H CRC. If
ated in all courses of sipuleucel-T, and cumula-
tion tended to be more robust in earlier disease
pronounced immune responses typical of MSI-H
vaccination with FSPs turns out to be well tolerated
tive CD54 upregulation was calculated for each
states.
CRC lesions may be explained by the generation
and leads to the induction of effector T-cell immune
patient. In some studies, cytokines were assayed
of defined MMR deficiency-induced antigens. MMR
responses, FSP vaccination might be considered as
from culture supernatants and T-cell and B cell ac-
deficiency causes insertion or deletion mutations at
a novel approach for tumor prevention in Lynch
tivation markers were assessed by flow cytometry
coding microsatellites, which may lead to shifts of
syndrome mutation carriers.
during manufacture.
the translational reading frame and to the genera-
Results: Baseline demographics were generally
tion of MMR deficiency-related frameshift peptides
representative of each disease state; neoadjuvant
(FSPs).
pts were younger with less disease burden; mCRPC
Using the resources of human genome databases
pts had the highest disease burden and more fre-
and a bioinformatics-based approach, we predicted
quent prior chemotherapy. While neoadjuvant pts
relevant target genes, which were then screened for
had greater levels of T-cells, the patterns of APC
mutations in a large set of MSI-H CRC specimens.
activation were similar among all trials, with in-
FSP sequences resulting from mutations at the most
creased CD54 upregulation at the second and third
frequently mutated coding microsatellite sequenc-
treatment. The trend for cumulative fold increase
es were then predicted and synthesized in vitro.
in CD54 upregulation was significantly greater in
Thirteen FSP antigens were evaluated in vitro and
earlier disease pts (neoadjuvant: 35.5, asymptomat221
168 | Therapeutic Vaccination
169 | Therapeutic Vaccination
Construction of DNA vaccines expressing the novel tumor
­antigen neuroplastin: protective efficacy against mammary
­adenocarcinoma in immunized mice
Dendritic-tumor cell hybrids in therapeutic vaccination against
advanced neuroblastoma
1
1
2
2
3
1
2
2
A. Tiptiri-Kourpeti , E. Poimenidis , P. Ypsilantis , C. Simopoulos , V. Schirrmacher 1
& K. Chlichlia Patrícia C. Bergami-Santos , Lilian Cristofani , Vicente Odone Filho ,
1
José Alexandre M. Barbuto 1
Laboratory of Immunobiology, Department of Molecular Biology and Genetics, Democritus
­University of Thrace, 68100 Alexandroupolis, Greece
1
Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, SP,
Brazil
2
Laboratory of Experimental Surgery and Surgical Research, Medical School, Democritus
­University of Thrace, 68100 Alexandroupolis, Greece
2
Department of Pediatric Oncology, Faculty of Medicine, University of São Paulo, SP, Brazil
Email: [email protected]
222
DNA vaccination targeting tumor antigens rep-
structs for two subsequent times resulted in strong
Neuroblastoma is the most common intra-abdomi-
and stored for use in vaccine preparation. Of all
resents an attractive technology to induce strong
neuroplastin-specific antibody responses in sera
nal tumor in children and, depending on partially
patients, 17 patients already received the vaccina-
antigen-specific immune responses as well as pro-
from immunized mice, as evidenced by indirect
established biological criteria, may have an ag-
tion. Nine patients received the treatment after au-
tective efficacy against a variety of tumors. Neuro-
ELISA using recombinant neuroplastin as antigen
gressive behavior and poor response to therapy.
tologous bone marrow transplantation (as included
plastin (NPTN) was recently identified as a poten-
and by immunofluorescence analysis. In addi-
For these cases, new therapeutic strategies are still
in the Brazilian neuroblastoma treatment protocol
tial tumor antigen using proteomic identification of
tion, immunization studies were performed using
needed. Monocyte-derived dendritic cells (Mo-
NEURO-X-2008). Clinical responses ranged from
affinity selected tumor proteins with a recombinant
a syngeneic experimental breast cancer model
DCs) can be a very powerful tool for the devel-
complete clinical remission to no response. The
variable heavy chain antibody. NPTN was found to
in mice. BALB/c mice were immunized with the
opment of immunotherapeutic strategies against
average number of doses received is 3.3 (one dose/
be highly expressed in the majority of metastat-
DNA vaccine constructs expressing neuroplastin
cancer. However, Mo-DCs from cancer patients
month) and no significant adverse side effects were
ic breast carcinomas and was shown to promote
for three subsequent times with an interval of 8-10
present a series of phenotypic and functional
noted. Thus, the further characterization of the re-
breast tumor growth and metastasis if aberrantly
days and challenged subcutaneously with synge-
changes that impair their potential to induce ef-
sponses induced by this treatment could establish
expressed. Therefore, we selected this antigen to
neic mammary adenocarcinoma cells. One week
fective anti-tumor responses. To circumvent these
this approach as an effective treatment modality for
design vaccine constructs aiming to be effective
later mice were killed and the tumor volume as
deficits, Mo-DCs from healthy donors can be fused
patients with neuroblastoma.
against mammary adenocarcinoma. We identified
well as tumor incidence was recorded. The majority
to patients’ tumor cells and, after irradiation, in-
the expression of NPTN gene in two murine adeno-
of pre- immunized mice showed no tumor growth
jected back into the patients to initiate anti-tumor
carcinoma cell lines and the complete cDNA se-
as opposed to the group of non-immunized mice or
responses. Fused cells maintain the expression
quence was cloned directly in mammalian expres-
mice immunized with the empty vector. Our aim is
of both tumor and Mo-DC markers for at least 7
sion vectors under the control of the CMV promoter.
to investigate and analyze further humoral and cel-
days after fusion. Furthermore, the heterokaryons
Two vaccine constructs were further examined, one
lular NPTN-specific anti-tumor immune responses
seem to survive and proliferate better than non-
of them encoding full-length NPTN, the other one
following different immunization strategies. Thus,
fused cells (both tumor and DCs) in culture. When
encoding the cDNA sequence of NPTN in fusion
we demonstrated that prophylactic immunization
fused cells were utilized to stimulate patients’ lym-
with the EGFP gene. Expression of NPTN was de-
with NPTN encoding DNA can induce protective
phocytes, they were able to induce the production
tected in both cases in vitro in NPTN-negative cell
immunity in mice against mammary adenocarci-
of a distinct cytokine pattern, characterized by a
lines transfected with the respective vaccine con-
noma and validated neuroplastin as a good target
higher IFN-gamma and a lower IL-4 production.
structs, as evidenced by Real-time PCR with NPTN
for cancer immunotherapy studies.
Interestingly, these fused cells induced a low pro-
specific primers as well as Western blotting with
liferative response on allogeneic lymphocytes. We
a neuroplastin-specific monoclonal antibody. Evi-
report further, the preliminary results of a protocol
dence was also provided for in vivo expression 48
using this hybrid dendritic-tumor cell vaccination
hours following intramuscular administration of
in patients with histological diagnosis of neuro-
the NPTN-expressing constructs. Moreover, intra
blastoma. Tumor samples from 63 patients were
ear pinna immunization of the DNA vaccine con-
obtained, processed into single cell suspensions
223
170 | Therapeutic Vaccination
171 | Therapeutic Vaccination
Active Immunotherapy of Cancer with PROSTVAC® and MVABN®-HER2 Demonstrate Potent Preclinical Anti-tumor Efficacy
Development of a DC-based therapeutic vaccine for AML
­patients: Charakterization of GMP-grade TLR-agonist matured
3-day DCs expressing the leukemia-associated antigens WT1
and PRAME
Stefanie J. Mandl, Ryan B. Rountree, Joseph Cote, Tracy dela Cruz, Thierry Giffon,
John R. Lombardo, Aung Oo, Erica Trent, Reiner Laus, Alain Delcayre
Department of Research and Development, BN ImmunoTherapeutics, Mountain View, CA, USA
S.J. Mandl and R.B. Rountree contributed equally to this work
224
*,1
*,2
*,3
2
Christiane Geiger , Barbara Beck , Iris Bigalke , Felix S. Lichtenegger , Mirjam H.M.
4
5
1, 3
2
Heemskerk , Stein Saboe-Larssen , Dolores J. Schendel , Marion Subklewe 1
Institute of Molecular Immunology and 3GMP Unit, Helmholtz Zentrum München, Munich,
Germany
2
Department of Internal Medicine III, University of Munich, Campus Großhadern, Munich, Germany
4
Department of Hematology, Leiden University Medical Center, Leiden, Netherlands
5
Department of Cellular Therapy, Oslo University Hospital, Oslo, Norway
*
Authors contributed equally to this work
BN ImmunoTherapeutics (BNIT) specializes in de-
mal growth factor receptor 2 (HER-2) that includes
We have designed a new generation of dendritic
lated cells – was analyzed in a so-called signal-3
veloping novel active immunotherapies for cancer.
two universal T-cell epitopes from tetanus toxin
cells (DCs) optimized for the use in cell-based im-
assay. We could show that antigen expression and
These therapies use recombinant poxviruses engi-
to facilitate the induction of effective immune re-
munotherapy of cancer patients. Our goal was to
cryopreservation did not alter this capacity.
neered to express tumor associated antigens (TAAs),
sponses against HER-2. Our Phase I clinical results
tailor these DCs to be used for vaccination in acute
Furthermore, cryopreserved DCs expressing the
with the intent of generating effective immune re-
show that HER-2-specific antibody and T-cell re-
myeloid leukemia (AML) patients with a high risk
different antigens also displayed a high capacity
sponses against the patients’ cancer. PROSTVAC®
sponses were induced in patients treated with
of relapse after intense induction/consolidation
both for reactivation of antigen-specific pre-primed
is a candidate product for the treatment of prostate
MVA-BN®-HER2.
therapy in order to eradicate minimal residual
effector cells and for priming of naïve T-cells in
cancer for which a global Phase III clinical trial
Here we show preclinical data demonstrating an-
disease (MRD).
vitro, showing proper processing and presentation
(PROSPECT) was recently initiated. This product
ti-tumor efficacy and TAA-specific antibody and
We have developed a three-day manufacturing
of the introduced antigens. These studies demon-
is composed of two different viral vectors derived
T-cell responses for both immunotherapies. Anti-
protocol using a cytokine cocktail containing a
strate that our manufacturing protocol yields im-
from a recombinant vaccinia virus (PROSTVAC®-V)
tumor activity was characterized by the induction
synthetic TLR7/8-agonist for generation of mono-
proved DCs with a high potential to initiate long-
and a recombinant fowlpox virus (PROSTVAC®-F).
of Th1-biased antigen-specific immune responses
cyte-derived mature DCs (mDCs) with improved
lasting anti-leukemic responses in patients with
Both vectors contain transgenes encoding prostate-
in preclinical HER-2-specific tumor models. Tumor
immunogenicity. For induction of a specific T-cell-
AML.
specific antigen (PSA) and a triad of costimulatory
efficacy was accompanied by increased infiltration
based anti-AML response against residual tumor
molecules (B7-1, ICAM-1, and LFA-3), designated as
of tumors with highly activated, HER-2-specific
cells, our mDCs are loaded with RNA encoding the
TRICOM™. Patients are immunized using a prime-
T-cells, and a decrease in the frequency of regula-
leukemia-associated antigens WT1 and PRAME.
boost strategy consisting of an initial treatment
tory T-cells (Treg). Likewise, treatment with PROS-
Additionally, DCs transfected with RNA encoding
with PROSTVAC®-V followed by repeated boosting
TVAC® results in strong efficacy in a mouse model
CMV-pp65 will be included as a surrogate antigen.
with PROSTVAC®-F to maximize the immune re-
of prostate cancer. Furthermore, the data illustrate
In this study, we present the careful evaluation of
sponses against the PSA tumor-antigen.
the advantage of the prime-boost strategy in induc-
our 3d mDCs generated from healthy donors follow-
Another product in development, MVA-BN®-HER2,
ing more robust immune responses against PSA.
ing RNA electroporation and cryopreservation, en-
is in Phase I clinical trials for the treatment of HER-
Overall, these studies demonstrate the potent activ-
suring a fully functional phenotype of the autolo-
2-positive breast cancer. This immunotherapy is
ity of poxviral immunotherapies in animal models
gous vaccine formulation. Our studies demonstrate
derived from a clonal isolate of the highly attenu-
of cancer, which supports the ongoing clinical de-
a high and controllable expression of all three anti-
ated Modified Vaccinia Ankara (MVA) virus stock
velopment of PROSTVAC® and MVA-BN®-HER2.
gens following RNA loading, which was also stably
known as MVA-BN®. The attenuated phenotype of
detected after cryopreservation. Additionally, ex-
MVA-BN® provides additional safety when given to
pression of common DC surface markers was not
immunocompromised patients, while providing a
altered by these processing steps. To ensure func-
strong adjuvant activity to transgenic antigens that
tional integrity of our DCs, the ability to secrete
triggers adaptive and innate immunity. MVA-BN®-
the critical cytokine IL12p70 upon T-cell encounter
HER2 expresses a modified form of human epider-
– an important characteristic of our non-manipu225
172 | Therapeutic Vaccination
173 | Therapeutic Vaccination
Dendritic cell vaccine after induction chemotherapy in patient
with metastatic melanoma: prospective randomized single-­
institution trial
A Novel Minigene Scaffold for Cancer Vaccine Applications
1
2
1
2
1, 2
3, 4
3, 4
5
3
Igor Samoylenko , Tatiana Zabotina , Irina Mikhailova , Olga Korotkova , Georgy ­Chkadua ,
4
4
1
1
Vladimir Tazaev , Elena Ogorodnikova , Kirill Baryshnikov and Lev Demidov 1
Takis, Rome, Italy
2
BIOGEM, scarl, via Camporeale, Ariano Irpino (Av), Italy
1
Department of tumor biotherapy, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia
3
2
Laboratory of clinical immunology, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia
Merck Research Laboratories, Rahway, NJ, USA
4
3
Laboratory of experimental diagnostics and tumor biotherapy, N.N. Blokhin Russian Cancer
Research Center, Moscow, Russia
Merck Research Laboratories, West Point, PA, USA
5
Okairos, Pomezia, Italy
6
Idera Pharmaceuticals, Cambridge, MA, USA;
7
IRCCS Istituto Nazionale Tumori Fondazione G. Pascale, Napoli, Italy
4
Department of blood transfusion, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia
Introduction: Despite the dramatic change in treatment
Results: From March 2010 to March 2012 95 patients
approaches in patients with metastatic melanoma and
were included in the study. After 2 cycles of chemo in
Cytotoxic T lymphocytes (CTL) represent a key
HLA-A0201 double transgenic (HHD) mice, the
implementation of new drugs during last few years
32 patients disease progression was detected and they
component of the immune system and play a crucial
latter being immunologically tolerant to CEA. The
the overall prognosis for survival remains poor. Rapid
were excluded from the study. Of the 63 patients in 3
role in tumor surveillance. CTLs recognize short
components utilized to construct the minigenes
disease progression often does not allow to register
patients effect was not assessed due to patient death
peptides derived from intracellularly synthesized
were: 1) epitopes selected on predicted binding to
any effects of immunotherapy. We decided to conduct
due to complications (1) and due to lost of follow up
proteins and expressed on surface of targeT-cells in
HLA-A0201, uniqueness in the human proteome
a vaccine trial in metastatic melanoma patients with a
(2). Twenty one patients are now under evaluation and
complex with MHC Class I (MHC-I) molecules. CTL
and increased likelihood of natural processing; 2)
stable disease course and to compare safety and effi-
were not included in this interim analysis. Of 39 pa-
epitopes have been identified in multiple clinically
helper CD4 epitopes; 3) epitope processing mecha-
cacy of the standard polychemotherapy with dendritic
tients 19 was randomized to the DC-group and 20 to
relevant tumor-associated antigens (TAA).
nisms; 4) fusion with immunoenhancing moieties.
cell-based autologous vaccine.
the chemo group. The 6-month PFS was 52,6% in the
Genetic vaccines utilize viral or plasmid DNA vectors
First, we show that minigenes delivered via
Aim: Primary end point was 6-month progression free
DC group and 15% in the chemo (RR = 0,56; 95% CI
to deliver in vivo an expression cassette carrying the
DNA-EP were more immunogenic than genetic
survival, secondary end points included overall sur-
0,34 to 0,97, p=0,03). Median PFS was 7,38 mo in the
coding region of the antigen of choice in the subject
vectors encoding the full-length protein or peptides
vival, progression free survival, safety, immunological
DC group and 4,9 mo in the chemo group (95% CI 6,3 to
to be vaccinated. Immunizations with minigenes
injected subcutaneously. In particular, we select an
parameters, and quality of life assessed by QLQ-C30
8,5 and 3,3 to 6,4 respectively, not significant). Median
containing T-cell epitopes may have several advan-
optimal minigene scaffold comprising the follow-
questionnaire.
OS was 13,4 mo in the chemo group and was not
tages compared to full length proteins. Proteins may
ing elements: human tissue plasminogen activator
Patients and methods: Inclusion criteria were morpho-
reached in the DC group. The most common grade 3-4
have unknown and potentially toxic biological ac-
(TPA) signal, T-cell epitopes and E. Coli enterotoxin
logically proven metastatic melanoma, ECOG status
adverse events were neutropenia (12 (60%) in chemo
tivity while minigenes deliver only immunologically
B subunit, wherein epitopes are connected by furin
0-2, LDH level <= 1,5N, RECIST-evaluable lesions,
group.0 in DC group), thrombocytopenia (3 (15%) and
relevant genetic information. Immunization with a
sensitive linkers. This family of minigenes was
skin or soft tissues lesions measurable by ultrasound.
1(5%)), febrile neutropenia (8 (20%) and 0), peripheral
protein usually leads to an immune response that is
also more immunogenic than a second class where
Any adjuvant treatment and single line of the meta-
sensor neuropathy (4 (20%) and 0), flu-like symptoms
narrowly focused on a few dominant epitopes, while
epitope processing is proteasome-dependent. The
static melanoma treatment were allowed. Patients with
(2 (10%) and 8 (42%)), tumor pain (2 (10%) and 3(16%)
minigenes can induce significant immune response
minigenes were able to break immune tolerance
brain metastasis were excluded. All patients scheduled
respectively). Scores of the QoL were better in the DC
to multiple epitopes simultaneously. Minigenes are
in CEA/HLA-A0201 mice and induce a strong
to receive 2 cycles of the polychemotherapy with cis-
group after the first cycle of the therapy than in chemo
short and compatible wth commonly used delivery
immune response against all included epitopes, in-
group at the corresponding time point. Immunological
vectors including plasmid DNA. Despite extensive
dependently of their relative positions within the
2
2
platin 20 mg/m day 1-4; vinblastin 2 mg/m day 1-4
2
+
hi
+
and DTIC 800 mg/m day 1, cycle 28 days. On day 14 of
testing suggested that population CD4 CD25 CD127+
studies with minigenes having different properties,
scaffold. These observations were extended also to
second cycle assessment was predefined. Patients who
(Tregs) did not predict therapy success and did not sig-
the provision of an optimal minigene that maximizes
other tumor antigens, such as HER2/neu and tel-
progressed after two cycles of the chemo were excluded
nificantly change during the treatment.
epitope-specific immune responses remains elusive.
omerase (TERT).
from the study. All other patients were randomized to
Conclusion: Dendritic cell vaccine immunotherapy
The objective of this study was to identify an optimal
In conclusion, here we describe a universal strat-
3-d cycle of the chemo followed by one vaccination
may bean less toxic option for maintenance therapy in
scaffold for minigene construction. To evaluate
egy to design minigenes delivered via DNA-EP and
cycle or three cycles of the chemo. Vaccination schedule
patients with metastatic melanoma with stable disease
the immunogenicity in an appropriate preclinical
based on predicted and/or experimentally identified
consisted of subcutaneous injections of dendritic cell
course. Final results will be published soon. Additional
model, we generated a set of minigenes contain-
epitopes. Further studies to evaluate this approach in
vaccine with 14 days intervals. Assessments were per-
trials are needed to compare such a vaccine with best
ing epitopes selected within the Carcinoembryonic
combination with other modalities, such as peptide
formed every 5 vaccine injections (1 cycle, 10 wks) in
supportive care or placebo.
Antigen (CEA) and utilized muscle DNA electropo-
vaccination or other genetic vectors are warranted.
DC-group and every 2 cycles (10 wks) in chemo group.
226
6
Luigi Aurisicchio , Arthur Fridman , Ansu Bagchi , Elisa Scarselli , Nicola La Monica ,
7
Gennaro Ciliberto ,
ration (DNA-EP) to vaccinate HLA-A0201 and CEA/
227
174 | Therapeutic Vaccination
175 | Therapeutic Vaccination
Generation of immunogenic MUC1 glycopeptides by DCs
primed with microvesicle bound MUC1 tumor associated glycoprotein, but not with the soluble MUC1
Tumor immunity conferred by mRNA-transfected dendritic
cells expressing bi-functional polypeptides which couple MHC-I
­presentation to dendritic cell activation
1
1
1
2
Hassan Rahimi , Chiara Napoletano , Federico Battisti , Francesca Belleudi , Ilaria Ziz1
1
3
4
2
zari , Valeria Visconti , Filippo Bellati , Hans Wandall , Maria Rosaria Torrisi , Marianna
1
1
Nuti , Aurelia Rughetti
1
Department of Experimental Medicine, “Sapienza” University of Rome, Rome, Italy
2
Department of Clinical and Molecular Medicine, “Sapienza” University of Rome, Rome, Italy
3
Department of Gynecology and Obstetrics, “Sapienza” University of Rome, Rome, Italy
4
Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen N, Denmark
2, 3
1
2, 3
1
& Lea Eisenbach
1
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
2
Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel
3
Department of Biotechnology, Tel-Hai College, Upper Galilee, Israel
Changes in glycosylation that occur during tumor
Two distinct MUC1 immunogens were employed:
Ex-vivo propagated, antigen-loaded dendritic cells
MHC-I peptide presentation to TLR-mediated sign-
transformation can profoundly affect the inter-
a recombinant soluble MUC1 produced by CHO-K1
(DCs) are widely explored as potential cancer vac-
aling and offer a safe, economical and highly ver-
actions between tumor cells and microenviron-
cells (ST-MUC1) and MUC1 membrane bound
cines, but the limited clinical efficacy achieved so
satile modality for a novel category of genetic CTL-
ment with strong impact on the overall anti-tumor
(MUC1-MV; microvesicles shed by the MUC1-trans-
far calls for novel approaches. Major obstacles are
inducing vaccines.
immune response.
fected DG75 cell line). Both MUC1 immunogens
the requirement for the induction of DC matura-
MUC1 is one of the most relevant tumor associ-
were enriched of ST carbohydrate moieties as de-
tion by adjuvants and the short duration of antigen
ated glycoprotein expressed by epithelial cancer.
tected by MALDI-TOF FS and by ELISA, employing
presentation. Recently we have generated a novel
Several tumor associated glycoforms of MUC1 are
anti-MUC1 glycoform specific antibodies (MoAbs).
genetic platform based on membrane-anchored
expressed by tumor cells and distinct effects on the
DCs were pulsed with ST-MUC1 and MUC1-MV
derivative of β2 microglobulin (β2m) linked to an
immune system have been described. In particu-
and reactivity of the distinct anti-MUC1 MoAbs
antigenic peptide of choice at its N-terminus and
lar the sialylated MUC1, carrying ST moieties (ST-
was analyzed by immunofluorescence microscopy
to the cytosolic domain of TLR4 at the C-termi-
MUC1) exerts inhibitory effects on DCs function,
on single optical sections obtained by Apotome
nus. Following mRNA transfection the resulting
while the MUC1 carrying N-AcetilGalactosamine
module. In DCs pulsed with the soluble ST-MUC1,
polypeptides efficiently coupled peptide presenta-
residues (Tn-MUC1) has shown to be immunogenic
only reactivity of the MoAb recognizing the sia-
tion to antigen-presenting cell activation. In the
in mice and to significantly correlates with tumor
lylated form of MUC1 could be detected. No reactiv-
present work we evaluated the immunogenicity
progression. The understanding of the intracellular
ity was found for the MoAbs recognizing the T and
of such constructs in mRNA-transfected murine
processing of TAA-MUC1 glycoprotein and how im-
immunogenic Tn-MUC1 glycoform. In DCs pulsed
bone marrow (BM)-derived DCs. We show that the
munogenic glycoepitopes can be generated is rel-
with the ST-MUC1 membrane bound form, that is
encoded peptide-β2m-TLR products are expressed
evant for the design of DCs based vaccine targeting
processed in HLAI and HLAII compartments, all
at the cell surface up to 96 hours post transfec-
MUC1 or other TAA-glycoproteins.
the MoAb against distinct MUC1 glycoforms were
tion, pair with endogenous heavy chains, prompt
We have previously shown that MUC1 undergoes
reactive, indicating that MUC1 processing was ac-
efficient peptide-specific T-cell recognition and
to distinct processing pathways in DCs as soluble
companied by deglycosylation, generating immu-
confer a constitutively activated phenotype on
molecule or bound to microvesicles.
nogenic Tn-MUC1 glycoepitopes. Similar results
the transfected cells. Ex-vivo mRNA-transfected
The soluble molecule, independently by the glyco-
were also obtained by biochemical analysis of cell
mouse BM-DCs were markedly superior in-vivo
sylation profile, appears to be blocked in the pre-
fraction of MUC1 pulsed DCs.
to peptide-loaded, LPS-activated DCs in inducing
endosomal compartment, while the membrane
These results strongly suggest that the antigen for-
peptide-specific CTLs. This superiority was evident
bound form is processed in HLAII and HLAI com-
mulation is of crucial importance both for cross-
in the ability to protect mice from tumor generation
partments. In this study we investigated the effect
processing and both for the generation of immuno-
following the administration of B16 melanoma cells
of antigen processing on carbohydrate moieties in
genic glycoepitope array when designing DC based
and to inhibit the development of pre-established
the generation of Tn-MUC1 glycoepitopes that have
vaccine.
B16 tumors. Our results provide evidence that the
been shown to be immunogenic.
228
1, 2
Gal Cafri , Alon Margalit , Esther Tzehoval , Gideon Gross
product of a single recombinant gene can couple
229
176 | Therapeutic Vaccination
177 | Therapeutic Vaccination
A First In Man Phase I Trial Of IMA950 (A Novel Multi-­Peptide Vaccine) Plus GM-CSF In Patients With Newly Diagnosed ­Glioblastoma –
Design And Preliminary Results of a Cancer Research UK Study
Induction of anti tumor responses against malignant
­melanoma via antigen targeting in vivo
2
3
4
5
Roy Rampling, Allan James , Paul Mulholland , Sharon Peoples , Omar Al-Salihi , Chris
6
7
8
8
9
9
Twelves , Sarah Jefferies , Oliver Schoor , Norbert Hilf , Jane Peters , Sarah Halford , Lesley
9
9
8
McGuigan , James Ritchie , Harpreet Singh-Jasuja
1
1
1
2
Kirsten Neubert , Anna-Maria Staedtler , Lukas Heger Veit Buchholz ,
1
3
1
Gordon F. Heidkamp , Falk Nimmerjahn , Diana Dudziak
1
Department of Dermatology, Laboratory of Dendritic Cell Biology, Friedrich-Alexander
­Universität Erlangen-Nuremberg, University Hospital of Erlangen, Erlangen, Germany
Beatson WOS Cancer Centre, Glasgow, UK
2
Department of Medical Microbiology and Hygiene, TU-München, Munich, Germany
3
UCL Cancer Institute, London, UK
3
4
Oncology Department, Western General Hospital, Edinburgh, UK
Chair of Genetics, University of Erlangen-Nuremberg, Laboratory of Experimental Immunology and Immunotherapy, Erlangen, Germany
5
Oncology Department, Southampton University Hospital, Southampton, UK
6
Oncology Department, St James University Hospital, Leeds, UK
7
Oncology Department, Addenbrookes Hospital, Cambridge, UK
8
immatics biotechnologies GmbH, Tubingen, Germany
9
Cancer Research UK Drug Development Office, London, United Kingdom
1
Department of Clinical Oncology, Glasgow University, Glasgow, UK
2
Background: Standard therapy for newly diag-
of two cohorts with similar schedules. In Cohort
Dendritic cells (DCs) as the most powerful antigen
results show that antigen targeting of DCs might be
nosed glioblastoma (NDGBM) comprises maximal
1 vaccination begins 7 to 14 days prior to initial
presenting cells (APCs) gaining increasing thera-
a future option for the induction of protective anti-
safe tumour resection, followed by concomitant
CRT; in Cohort 2 it begins a minimum of 7 days
peutic significance. Because of their role as key
tumor responses.
chemoradiotherapy (CRT) and adjuvant temozo-
post CRT and 28 days prior to adjuvant TMZ. Safety
regulators for the coordination and balance of the
lomide (TMZ). IMA950 is a novel multi-peptide
is assessed according to NCI CTCAE Version 4.0.
innate and acquired immune response they are
GBM-specific vaccine that contains 11 HLA-bind-
Immune response is determined by HLA-multim-
good candidates to modulate the immune system
ing tumour-associated peptides (TUMAPs), which
er analysis of vaccine-induced T-cell response in
activity in infection, cancer and autoimmunity.
were identified on human leukocyte antigen (HLA)
PBMC samples. Secondary objectives include ob-
By using chimeric antigen carrying antibod-
surface receptors in primary human GBM tissue,
servation of any anti-tumour effects, measurement
ies directed against the DC-subset specific C-type
and one viral (HBV) marker peptide. The selected
of pre-treatment regulatory T-cell levels and evalu-
lectin and endocytosis receptors DCIR2 (33D1) and
TUMAPs are designed to activate TUMAP-specific
ation of the effect of steroid dose on observed T-cell
DEC205, we are able to target antigens to CD11c CD8
+
+
-
+
CD8 cytotoxic and CD4 helper T lymphocytes,
responses. Retrospective analysis of diffusion and
or CD11c CD8 DCs in vivo, respectively. We have
which then recognise cognate TUMAPs presented
perfusion-weighted imaging is being performed to
demonstrated that the type of T-cell response gener-
by GBM tumour cells and effect a targeted immune
explore possible vaccination effects. Patients will
ated is dependent on the DC subset that presents the
response. The primary objectives of the current
also be followed up for overall survival.
antigen in vivo. Here, we wanted to investigate if we
study are to assess the safety, tolerability and im-
Results: As of 29-Mar-12, 22 patients (11 in Cohort
can induce a protective anti-melanoma response by
munogenicity of IMA950 plus GM-CSF given along-
1 and 11 in Cohort 2) have been recruited. Eight-
targeting DCs in naïve animals in vivo. For induc-
side standard therapy in NDGBM.
een remain alive. Related adverse events have been
ing an efficient immune response antigen carrying
Methods: Patients must be eligible for standard
restricted to minor injection site reactions and a
antibodies 33D1 or DEC205 were applied under im-
treatment of GBM, and HLA-A*02 positive with
single distant allergic rash. The first 6 patients
munizing conditions. In the used murine melanoma
no history of autoimmune disease. Vaccination
have been fully analysed for immune response;
mouse model and the used immunization protocol
comprises fixed doses of IMA950 plus GM-CSF
all reacted positive for HBV peptide. Five subjects
mice showed a mixed TH1/TH2 mediated antibody
injected intradermally at 11 time points over a
have responded to at least one TUMAP, 3 of these
response and a strongly prolonged survival with a
24 week period. Up to 45 patients with NDGBM
to multiple TUMAPs.
diminished tumor growth. Moreover, antigen target-
will be entered. Three safety observation periods
Conclusion: These results vindicate the study
ing to both DC subsets induced an even better anti
of 21 days were included after patients 1, 3 and 6
design and already give encouragement for further
tumor response. Antigen targeting in a therapeutic
had completed treatment and prior to opening to
development of this vaccine.
setting which is clinical more relevant induced a
general recruitment. Patients are recruited into one
230
+
+
This project was partly supported by the German
Research Foundation (DU548/1-1 and DU548/2-1), GIF
(2177-1774.11/2007), Ria-Freifrau-von Fritsch Stiftung
and BayGene. D.D. is a fellow of the ‘Förderkolleg’ of the
­Bavarian Academy of Sciences.
delayed tumor growth and prolonged survival. Our
231
178 | Therapeutic Vaccination
179 | Therapeutic Vaccination
Comparison of clinical grade polarized and standard matured
dendritic cells for cancer immunotherapy
Investigating the functionality of tumour-infiltrating
­lymphocytes induced by immunotherapy
1
2
1
2
1
Morten Hansen , Gertrud Hjortø , Özcan Met , Niels Bent Larsen , Mads Hald Andersen ,
1
3
1, 4
Per thor Straten , Pawel Kalinski and Inge Marie Svane
Elena Harden, Angelica Cazaly, Christian Ottensmeier and Stephen Thirdborough
Faculty of Medicine, University of Southampton, Southampton, SO16 6YD, UK.
1
Center for Cancer Immune Therapy (CCIT), Department of Haematology, Copenhagen
­University Hospital, Herlev, Denmark
2
Department of Micro- and Nanotechnology, Technical University of Denmark, Lyngby, Denmark
3
Department of Surgery, Department of Immunology and Department of Infectious Diseases
and Microbiology, University of Pittsburgh, Pittsburgh, PA 15213; and University of Pittsburgh
Cancer Institute, Pittsburgh, PA, 15213, USA
4
Department of Oncology, Copenhagen University Hospital, Herlev, Denmark
Monocyte-derived dendritic cells (DCs) employed
equal to sDCs. mpDCs were intermediate between
The TRAMP (transgenic adenocarcinoma of the
which we have also found indications of immuno-
for cancer immunotherapy are commonly matured
standard and polarized DCs both in terms of IL-12
mouse prostate) mouse is a model of prostate
suppression in TILs.
by a standard maturation cocktail consisting of
secretion and ability to migrate. However, strik-
cancer. These mice express the large T antigen
These results indicate that CD8 T-cells induced by
inflammatory cytokines (TNF-α, IL-1β, IL-6) and
ingly mpDCs expressed significantly more of the
from SV40 under the control of a prostate-specific
DNA vaccination are subject to local immunosup-
prostaglandin E2. The major limitation of this cock-
immune-inhibitory molecules PD-L1 and CD25
promoter, resulting in spontaneously occurring
pression in the tumour microenvironment. Combi-
tail is the lack of Toll-like receptor mediated activa-
compared with standard DCs. Thus, despite their
prostate carcinomas in males. We have used this
national therapy with immune-modulating agents
tion that in turn does not enable DCs to produce
ability to produce some IL-12, they are likely not an
model to study DNA vaccination as immunother-
may be able to overcome this suppression and allow
the third signal pro-inflammatory cytokine IL-12
optimal replacement of the current gold standard
apy for cancer.
CD8 T-cell mediated tumour destruction. This in-
and subsequent polarize T-cells into type 1 effector
for DC maturation.
The DNA vaccine encodes tumour-derived epitopes
formation will be crucial when planning the design
cells that can be clonally expanded and generate
fused to a domain from tetanus toxin (DOM). This
of future DNA vaccine clinical trials.
memory. Here, standard matured DCs were com-
microbial sequence induces T-cell help in tandem
pared with DCs matured with either of two type
with the CD8 T-cells induced by the tumour-spe-
1 polarizing maturation cocktails namely “aDC1s”
cific part of the vaccine. This pDOM-epitope design
(TNF-α, IL-1β, IFN-γ, IFN-α, PolyI:C) and “mDCs”
has been shown to break immunological tolerance
(MPLA, IFN-γ) or a mixed cocktail – “mpDCs”, com-
and produce anti-tumour immune responses that
prising MPLA, IFN-γ and PGE2. Monocytes were ob-
prolong patient survival.
tained by elutriation of leukapheresis samples from
While tumour growth in TRAMP mice was delayed
six healthy donors and DCs were produced in large
by DNA vaccination, it was still fatal. In order to in-
scale with serum free CellGenix resembling clinical
vestigate this further we have isolated T-cells from
protocols. aDC1s and mDCs secreted >10 ng/ml of
the blood and the tumour and tested their function-
IL-12 after 48 hours of maturation and maintained
ality. The DNA vaccine was able to induce CD8
secretion following 24 hours of re-stimulation with
T-cell responses in mice with substantial tumour
CD40L-expressing J558 cells. Furthermore, aDC1s
load. Furthermore, greater numbers of tetramer-
and mDCs were superior in their ability to polar-
positive CD8
+
232
+
+
+
+
+
T-cells were found in the tumour
ize naïve CD4 T-cells into IFN-γ Th1 effector cells
compared to the blood. However, despite the high
but were less capable of CCL21-directed transwell
proportion of tetramer-positive tumour-infiltrating
migration compared with sDCs. This was likely
lymphocytes (TILs), effector cytokine production
due to a proportional increased ability to secrete
amongst TILs was low. In addition, TILs expressed
CCL19, mediating transient internalization of CCR7.
the marker PD-1 which is associated with function-
Upon 24 hours of re-culture in new medium, type
al exhaustion. These findings are mirrored by pre-
1 polarized DCs re-gained their ability to migrate
liminary data from immunotherapy trial patients in
233
180 | Therapeutic Vaccination
Study of the involvement of the activity of oxygen free radicals
in the development of colorectal cancer: chemo-preventive effect
of an antioxidant SOD mimetic a Glisodin®
Ouali Kheireddine, Baba-Ahmed Fedia, Trea Faouzia
University of Annaba, Faculty of sciences, BP 12 El Hadjar Annaba 23000, Algeria
Tumor Biology and Interaction
with the Immune System
This study was conducted to assess the effectiveness of supplementation of a vegetal SOD mimetic
a Glisodin on the number of precancerous lesions
of the aberrant crypt focus (ACF), and oxidative
status (lipid peroxidation, the antioxidant defense
system) in animal model with colorectal cancer
induced chemically by azoxymethane. In fact, administration of azoxymetomhane (AOM) caused
by the appearance of precancerous lesions of the
colon revealed by the formation of aberrant crypt
foci (ACF) are preneoplastic lesions. This cytological alteration colon goes along with increased lipid
peroxidation (LPO) and a significant decrease in
reduced glutathione (GSH), glutathione - S - transferase (GST), superoxide dismutase (SOD) and
catalase (CAT), which are potential biomarkers of
oxidative stress. Treatment of AOM rats by Glisodin
has significantly strengthened antioxidant defense,
showed an improvement in the activity of GST,
SOD and CAT, and decreased lipid peroxidation.
This effect had a positive impact on the number
of aberrant crypt foci. Our results suggests both
that the increase production of free radicals may
be induced a formation of precancerous lesions and
on the other hand, Glisodin can be used as an antiradical adjuvant with chemo-preventive against
colon cancer.
Keywords: AOM, Oxidative stress, Colorectal cancer, ACF,
Rat
235
234
181 | Tumor Biology and Interaction with the Immune System
182 | Tumor Biology and Interaction with the Immune System
The effects of ADAM10 and Neprilysin on tumor-induced
­release of IL-6 and IL-10
The Immunomodulatory Role of Endogenous Glucocorticoids
in Ovarian Cancer
Nuray Erin, Nilüfer Ekinci, Elie Cope, Ismat Khatri, Reg Gorczynski
Ahmed Adel Seida , Sebastian Häusler , Claudia Heidbrink , Jörg Wischhusen
Akdeniz University School of Medicine, Dept. Medical Pharmacology, SBAUM, Antalya and
­University Health Network, Toronto, ON
1
Department of Obstetrics and Gynecology, University of Würzburg, School of Medicine,
­Josef-Schneider-Street 4, 97080 Würzburg, Germany
2
Interdisciplinary Center for Clinical Research, Josef-Schneider-Street 2, 97080 Würzburg, Germany
236
1, 2
1, 2
1
1, 2
There are controversial findings regarding the role
induced by 4THM cells but IL-10 secretion induced
Background: Tumour-infiltrating myeloid-derived
glucocorticoids by immune cells may contribute to
of proteases in cancer progression. We previously
by LPS was decreased markedly only in NEP-treat-
suppressor cells (MDSC) or tumour-associated mac-
the immune escape of ovarian cancer is now being
found that expression of ADAM10, a member of
ed samples. Similar changes were also observed in
rophages (TAM) which are abundant in ovarian
tested in PTEN
matrix metalloprotease and disintegrin family, de-
MLCs from control animals. Importantly, neither
cancer show a high expression of the enzyme 11Be-
which spontaneously develop ovarian cancer after
creases in 4THM primary tumors which is a more
ADAM10 nor NEP hydrolyzed either cytokine
ta-Hydroxysteroid dehydrogenase I (11β-HSD1).
intra-bursal injection of adenoviral Cre recom-
metastatic subset of 4T1 murine breast carcinoma.
under in vitro conditions.
loxP/loxP
; loxP-Stop-loxP-kras
G12D
mice
This enzyme is essential for the conversion of
binase. The ongoing experiments involve adop-
We have also reported that ADAM10 has similar
biologically inactive cortisone into active cortisol
tive transfer of glucocorticoid receptor knock out
substrate specificity to Neprilysin (NEP), a pepti-
which has been detected in ascitic fluid and tumour
immune cells as well as pharmacological inhibition
dase reported to regulate immune response as well
exudates from ovarian cancer patients. Consider-
of 11β-HSD1 which shall be combined with various
as inhibit tumor growth. IL-10 and IL-6 may also
ing that cortisol has strong suppressive effects on
immune stimuli. In a first functional in vivo assay,
enhance tumor growth and metastasis, the former
all kinds of immune cells, we hypothesize that the
the adoptive transfer of glucocorticoid receptor-de-
through its immunosuppressive effects, the latter
activation of endogenous glucocorticoids by MDSC
ficient T-cells led to increased immune cell infiltra-
by inducing excessive inflammation. How these
or TAM may contribute to the immune escape of
tion of the tumour tissue – which did not translate
cytokines are regulated by ADAM10 and/or NEP
ovarian cancer. Material and methods: Using im-
into prolonged survival. Instead, infiltrating T-cells
is unknown. The goal of the present study was to
munohistochemistry, real-time PCR, luminescent
assumed mostly a Foxp3 (regulatory) phenotype
evaluate the effects of ADAM10 and NEP on tumor-
immunoassays (LIA), immunofluorescent double
and survival was even shortened.
induced release of these cytokines using mix leuko-
staining and adoptive transfer of glucocorticoid
Conclusion: We thus propose that endogenous glu-
cyte cultures (MLCs).
receptor knock out immune cells into immune de-
cocorticoids exert immunomodulatory functions
MLCs were initiated using spleen and peripheral
ficient mouse model for ovarian cancer.
in ovarian cancer. Their putative role in tumour
lymph nodes of female Balb/c mice injected with
Results: We found that 11β-HSD1 enzyme is highly
immune escape, however, needs to be assessed
4THM cells orthotopically 12 days earlier. Control
expressed in human and murine ovarian cancer
in context of further tolerogenic mechanisms that
MLCs used cells from non-injected females. MLCs
tissue. Luminescent immunoassays (LIA) showed
may be simultaneously present.
were stimulated with either tumor cells or LPS.
elevated cortisol levels in serum, ascites and tissue
A control group contained unstimulated cells.
exudates from ovarian cancer patients as com-
ADAM10 or NEP was added to MLCs at 10ng/ml
pared to healthy controls. Immunofluorescent
concentration. Changes in cytokine levels in the
double staining revealed a co-localization of 11β-
culture supernatant were determined at 40 hours
HSD1 with CD14, CD68, and CD85, but not with
by ELISA.
EpCAM. Expression of 11β-HSD1 can thus be at-
Results: Both ADAM10 and NEP significantly de-
tributed to tumour associated macrophages (TAM)
creased IL-6 secretion induced by stimulation of
or myeloid derived suppressor cells (MDSC).To test
MLC with 4THM cells. IL-10 secretion was not
our hypothesis about activation of endogenous
+
237
183 | Tumor Biology and Interaction with the Immune System
184 | Tumor Biology and Interaction with the Immune System
Characterization of breast cancer stem cells and their
­correlation with circulating tumor cells
Extracellular adenosine metabolism mediated by myeloid
­derived suppressor cells in melanoma and pancreatic cancer
1
1
1
1
1, 2
Siripakorn Sangkitporn , Somchai Sangkitporn , Patcharaporn Boonchu , Chonlada Yodtup ,
2
Kris Chatamra
1
Clinical Research Center, Department of Medical Sciences, Nonthaburi, Thailand
2
Queen Sirikit Centre for Breast Cancer, Chulalongkorn Hospital, Bangkok, Thailand
1
1
German Cancer Research Center and University Hospital Mannheim, Heidelberg, Germany
2
Department of General Surgery, University of Heidelberg, Heidelberg, Germany
Recent studies suggest that cancer stem cells
3D culture within 3 days. Flow cytometry results
Extracellular ATP and adenosine have recently
Moreover, CD73 levels were markedly higher on
(CSCs) are responsible for tumor initiation, inva-
show that phenotype of CSCs in both primary cell
emerged as important signaling molecules acti-
granulocytic MDSC. Most importantly, numbers
sion, metastasis and resistance to anticancer thera-
cultures and EDTA blood samples carried CD44 /
vating and suppressing the anti-tumor immune
of both granulocytic and monocytic CD73 MDSC
+
-
-
+
pies. The connection of CSCs to circulating tumor
CD24 / CD45 / EpCAM-. Molecular characteriza-
response, respectively. The stepwise conversion
were strongly increased in tumor-bearing mice.
cells (CTCs) is complex and currently under debate.
tion results demonstrate that Her2, ER, Mucin1
of ATP into adenosine by ectonucleotidases CD39
Therefore, enhanced adenosine production may
CTCs are easily to obtain by peripheral blood sam-
and actin genetic markers were positive whereas
and CD73 provides a robust mechanism of immune
contribute to MDSC-mediated suppression of anti-
pling, which can be repeated frequently, allowing
EpCAM was negative.
regulation. Ectonucleotidase expression and their
tumor response by MDSC.
real-time monitoring of metastatic progression.
Our findings demonstrate that CSCs that express
activity in cancer cells and tumor infiltrating lym-
Based on these data, we presumed that inhibition
Currently, CTCs are being integrated into clinical
CD44 / CD24 were disseminated from the primary
phocytes have been linked to the tumor associated
of adenosine production and/or signaling might di-
trial design as surrogate markers in development
tumor into the circulation. The characterization
immune suppression. However, the mechanisms
minish MDSC immunosuppressive functions and
of targeted therapies.
and enumeration of CSCs in peripheral blood
modulating adenosine production by immune cells
lead to a positive clinical effect. To this end, we
In order to study the correlation between CSCs and
samples could be used as liquid biopsy whereby
remain largely unknown.
treated ret transgenic tumor-bearing mice with a
CTCs, phenotype and genotype characteristics of
a simple test with minimal risk will permit tumor
In the current study, we investigated the role of
CSCs separated from primary cultures of breast
characterization; provide real time information
ectonucleotidases on CD11b Gr1 myeloid derived
antagonist (SCH58261). Indeed, these therapies
cancer tissues were compared with those of CTCs.
about the patient’s current disease state, identifica-
suppressor cells (MDSC) in mouse models of mela-
resulted in a significantly prolonged survival of
Tumor tissues and EDTA blood samples were col-
tion of treatment targets and aid in selection of the
noma and pancreatic adenocarcinoma. Ret trans-
tumor-bearing animals.
lected from invasive ductal carcinoma patients.
most appropriate targeted therapy.
+
-
+
+
CD73 inhibitor (APCP) or adenosine A2a receptor
genic mice were used as a spontaneous melanoma
Taken together, our data suggest that therapeutic
Tumor tissues were processed based on mechani-
model, which closely resembles human melanoma.
inhibition of adenosine production and signaling
cal disaggregation followed by enzyme digestion.
In the orthotopic model of pancreatic ductal adeno-
targets an important mechanism of immunosup-
Cancer cell suspensions were grown in 2D mon-
carcinoma, highly tumorigenic Panc02 cells were
pression and holds promise as an efficient strategy
olayer and 3D tumor sphere cultures. CTCs in EDTA
injected directly into the head of pancreas of im-
to boost an anti-tumor immunity.
blood samples were enriched by density gradient
munocompetent mice.
centrifugation. CD24, CD44, CD45 and EpCAM ex-
We found that CD39 is constitutively expressed on
pression as surface markers were determined by
all CD11b Gr1 cells in healthy and tumor-bear-
multi-parameter flow cytometry. Genetic markers
ing animals. This implies the capability of MDSC
including Her2, ER, Mucin 1, EpCAM and internal
to hydrolyze proinflammatory ATP into AMP.
control actin were detected by RT-PCR.
In contrast, the distribution of CD73 was highly
In 2D culture, tumor cells were very pleomor-
heterogeneous. In particular, the granulocytic
phic. Most commonly the cells were triangular
(CD11b Ly6C
or spindle-shaped. Multinucleate cells were ob-
significantly higher number of CD73 cells than the
served. Tumor cells could form tumor spheres in
238
2
Ivan Shevchenko , Alexandr V. Bazhin and Viktor Umansky
+
+
+
-/low
+
Ly6G ) MDSC subset contained a
+
+
monocytic subpopulation (CD11b Ly6C
high
-
Ly6G ).
239
185 | Tumor Biology and Interaction with the Immune System
186 | Tumor Biology and Interaction with the Immune System
Development of Resistance towards Artesunate in MDAMB-231
Human Breast Cancer Cells Treatment
Characterization of dormant melanoma cells and their
­interaction with memory CD8 T-cells in ret transgenic mouse
melanoma model
1, 2
3
1
1
2
Beatrice Bachmeier , Iduna Fichtner , Peter H. Killian , Emanuel Kronski , Ulrich Pfeffer ,
4*
Thomas Efferth
Fernando Flores-Guzman, and Viktor Umansky
German Cancer Research Center (DKFZ) and University Hospital Mannheim, Heidelberg, Germany
1
Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University,
Munich, Germany
2
Functional Genomics, Advanced Biotechnology Center, Genoa, Italy
3
Department of Experimental Pharmacology, Max Delbrück-Center for Molecular Medicine,
Berlin, Germany
4
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,
Johannes Gutenberg University, Mainz, Germany
*
E-mail: [email protected]
+
(2 and 4 TRP-2 CD133
that neoplastic clones are maintained by a rare frac-
cells respectively). We confirmed the dormant
tion of tumor cells with stem cell properties. CSC
state of TRP-2 CD133
could represent disseminated dormant tumor cells
the absence of Ki67 and PCNA expression. p16 and
+
+
cells /106 bone marrow
melanoma cells reflected
without clinical signs of tumor progression. We
p27, which typically are located in the nuclei of
Breast cancer is the most common cancer and the
efficiently abolished as compared to the control
used a ret transgenic mouse spontaneous melano-
dormanT-cells, were found in the cytoplasmic com-
second leading cause of cancer death in industrial-
drug doxorubicin. Taken together our in vitro and
ma model, in which 25% of transgenic mice develop
partment of TRP-2 CD133 melanoma cells indicat-
ized countries. Systemic treatment of breast cancer
in vivo results correlate well showing for the first
skin tumors with metastases in lymph nodes, liver,
ing their highly malignant phenotype. Investigat-
is effective at the beginning of therapy. However,
time that artesunate induces resistance in highly
brain and lungs. Mice without macroscopic tumors
ing the interaction between memory CD8 T-cells
after a variable period of time, progression occurs
metastatic breast tumors.
older than 20 weeks contain in the bone marrow
with disseminated melanoma cells in the bone
tyrosinase related protein (TRP)-2-specific effector
marrow, we found that TRP-2 Ki67
due to therapy resistance. Artesunate, clinically
+
+
+
+
neg
melanoma
+
used as anti-malarial agent, has recently revealed
Selected references:
memory CD8 T-cells and show no further melano-
cells were co-localized with memory CD8 T-cells
remarkable anti-tumor activity offering a role as
Efferth et al. Molecular Pharmacology 2003;64:382-94.
ma progression. This suggests a potential role of
both in tumor free and tumor bearing mice. The
novel candidate for cancer chemotherapy. We ana-
Dell’Eva et al. Biochemical Pharmacology 2004;68:2359-6.
dormant tumor cells in the maintenance of memory
proportion of memory CD8 T-cells interacting with
lyzed the anti-tumor effects of artesunate in me-
Efferth. Drug Resistance Updates 2005;8:85-97.
CD8 T-cells. We found that TRP-2 CD133 melano-
TRP-2 Ki67
tastasizing breast carcinoma in vitro and in vivo.
Efferth. Current Drug Targets 2006;7:407-21.
ma cells represent <1.2% in primary skin tumors,
15%) in the bone marrow of these mice. Quantita-
Unlike as expected, artesunate induced resistance
Kelter et al. PLoS One 2007;2:e798.
metastatic lymph nodes, and bone marrow metas-
tive analyses revealed that although certain IFN-γ-
in highly metastatic human breast cancer cells
Efferth et al. PLoS One 2007;2:e693.
tases. The majority of these cells were Ki67-negative
producing CD8 T-cells interacted either with single
MDA-MB-231. Likewise acquired resistance led to
Efferth et al. Molecular Cancer Therapeutics 2008;7:152-61.
suggesting thereby that these cells could remain in a
TRP-2 melanoma cells or the smallest cluster of
abolishment of apoptosis and cytotoxicity in pre-
Li et al. Cancer Research 2008;68:4347-51.
dormant state. We found an increased expression of
melanoma cells (2-5 TRP-2 melanoma cells), none
treated MDA-MB-231 cells. In contrast, artesunate
Bachmeier et al., PLoS One 2011;6:e20550
HIF-1a on TRP-2 CD133 melanoma stem-like cells
of these cells could produce perforin. Only two
in large tumors in comparison with those in smaller
TRP-2-specific CD8 T-cells were able to produce
was more cytotoxic towards the less tumorigenic
+
+
+
+
+
+
+
+
+
neg
melanoma cells was lower (less than
+
+
+
+
MDA-MB-468 cells without showing resistance.
tumors. Interestingly, TRP-2 CD133 HIF-1a mela-
perforin, but none were co-localized neither with
Unraveling the underlying molecular mechanisms,
noma stem-like cell fraction had a higher capacity to
TRP-2 melanoma cells nor TRP-2 CD133 melano-
we found that resistance was induced due to acti-
proliferative in smaller tumors in comparison with
ma stem-like cell in tumor free and bearing mice.
vation of the tumor progression related transcrip-
those in large tumors. To investigate whether TRP-
Furthermore, memory CD8 T-cells embedded into
+
+
+
+
+
+
+
tion factors NF-κB and AP-1. Thereby transcription,
2 CD133 melanoma cells are disseminated in the
large clusters of melanoma cells (50 TRP-2 tumor
expression and activity of the matrix-degrading
bone marrow of ret transgenic mice, we performed
cells) were unable to produce perforin and IFN-γ.
enzyme MMP-1, whose function is correlated with
an immunofluorescence. We found that only 40%
These findings suggest that dormant TRP-2 tumor
increased invasion and metastasis, was up-regulat-
of mice without macroscopic tumors (n=20) con-
cells might maintain memory CD8 T-cells. In con-
+
+
+
+
ed upon acquisition of resistance. Additionally, ac-
tained TRP-2 CD133 melanoma cells in the bone
clusion, our data demonstrate an existence of the
tivation of the apoptosis-related factor NF-κB lead
marrow. In contrast, all tumor bearing mice (n=20)
subpopulation of CD133
to increased expression of ant-apoptotic bcl2 and
240
+
The hypothesis of cancer stem cell (CSC) suggests
+
+
+
+
contained TRP-2 CD133
melanoma cells. The
reduced expression of pro-apoptotic bax. Applica-
amount of TRP-2 CD133
melanoma cells in the
tion of artesunate in vivo in a model of xenografted
bone marrow of mice without macroscopic tumor
breast cancer showed, that tumors growth was not
was significantly lower than in tumor bearing mice
+
melanoma cells in ret
+
transgenic mice. Dormant TRP-2 melanoma cells
+
are able to interact with CD8 T-cells in the bone
marrow of tumor-bearing mice.
241
187 | Tumor Biology and Interaction with the Immune System
188 | Tumor Biology and Interaction with the Immune System
Cyclophosphamide-induced myeloid-derived suppressor cells:
their functional characterization and modulation by
­5-azacytidine and IL-12
TLR3-expressing Tumor Parenchyma and Infiltrating NK Cells
Promote Tumor Control in Hepatocellular Carcinoma Patients
Romana Mikyšková, Marie Indrová, Veronika Polláková, Jana Bieblová, Jana Šímová and
Milan Reiniš
Valerie Chew, Charlene Tow, Emilie Bard-Chapeau, Neal G. Copeland, Nancy A. ­Jenkins,
3
4
5
6, 7
8
Achim Weber, Kiat Hon Lim, Han Chong Toh, Mathias Heikenwalder, Irene Ng,
1
1
­Alessandra Nardin, Jean-Pierre Abastado
1
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech
Republic
2
2
2
1
Singapore Immunology Network (SIgN), Agency for Science, Technology and Research
(A*STAR), Biopolis, Singapore
2
Institute Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR),
Biopolis, Singapore
Myeloid-derived suppressor cells (MDSC) are
ability of CY-MDSC did not reach that of TU-MDSC.
3
Department of Clinical Pathology, University Hospital of Zurich, Switzerland
major contributors to the mechanisms responsible
In order to mimic the clinically relevant setting, a
4
for tumour-induced immunosuppression. MDSC
group of mice bearing TC-1 tumours treated with
Department of Pathology, Singapore General Hospital, Singapore
5
represent a heterogeneous population of undif-
CY was also included into this comparison. The
Department of Medical Oncology, National Cancer Centre, Singapore
ferentiated myeloid cells, broadly defined in mice
phenotype and function of MDSC obtained from
6
Institute of Neuropathology, University Hospital Zurich, Switzerland
Institute of Virology, Technical University München / Helmholtz Zentrum München, Germany
Department of Pathology, The University of Hong Kong, Queen Mary Hospital, and State Key
Laboratory for Liver Research, The University of Hong Kong, Hong Kong
as CD11b Gr-1 cells. The main feature of these
the CY-treated, TC-1 tumour-bearing mice were, in
7
cells is their ability to suppress T-cell responses.
general, found to lie between CY-MDSC and TU-
8
MDSC accumulate in lymphoid organs and blood
MDSC.
under different pathologic conditions, e.g. tumour
Intraperitoneal or peritumoral treatment with
growth, infection, or inflammation. MDSC mobi-
5-AZA decreased the percentage of MDSC in the
lization was also reported after administration of
spleens of TC-1 tumour-bearing mice. A decrease in
widely used antineoplastic drug, DNA alkylating
the percentage of MDSC was also noticed in tumour
Background: Hepatocellular Carcinoma (HCC) is
ing T and NK cells and 4) enhanced apoptosis with
agent, cyclophosphamide (CY).
microenvironment and this was accompanied by
a highly aggressive cancer linked to chronically
reduced proliferation of tumor parenchyma cells in
The aim of our experiments was to characterize
a decrease of arginase-1, one of the mechanisms
dysregulated liver inflammation. However, appro-
vivo. Accordingly, TLR3 expression in human HCC
+
the phenotype and function of spleen CD11b /Gr-1
by which MDSC exert their function. 5-AZA was
priate immune responses can control HCC progres-
patients correlated with NK cell activation, NK and
cells that accumulate after CY therapy (CY-MDSC)
also able to decrease the percentage of MDSC in the
sion. Here we investigated the role of TLR3 in HCC
T-cell infiltration into tumors, and with decreased
and to compare them to those expanded in mice
spleens of mice that underwent CY administration.
patient survival and the underlying mechanisms.
viability of tumor parenchyma cells.
bearing HPV16-associated murine TC-1 carcinoma
After in vitro cultivation of MDSC in the presence
Methods: HCC cell death, NK cell activation and
Conclusion: Taken together, these data demon-
(TU-MDSC). Further, we evaluated whether this
of IL-12, the percentage of CD11b /Gr-1 cells de-
cytotoxicity were assessed in vitro after treatment
strate that TLR3 is an important modulator of HCC
population could be affected with DNA methyl-
+
creased and the percentage of CD86 /MHCII
with the TLR3 ligand poly(I:C). The role of TLR3
progression and represents a potential target for
transferase inhibitor 5-azacytidine (5-AZA) or with
cells increased. This effect was accompanied by
on the tumor parenchyma and infiltrating immune
novel immunotherapy.
interleukin 12 (IL-12), a potent immunostimulatory
a decrease in the relative expression of immuno-
cells was also investigated in a spontaneous tumor
cytokine with the ability to differentiate MDSC into
suppressive genes and lower VEGF production.
mouse model (n=6 mice per group). These results
antigen-presenting cells.
A stronger modulatory effect was noticed in the
were validated using tumor samples from 172 HCC
Although both CY-MDSC and TU-MDSC acceler-
group of CY-MDSC.
patients. Survival was analyzed by Kaplan-Meier
ated growth of TC-1 tumours in vivo, their phe-
Our findings identified similarities and differences
method using log-rank test and all statistical tests
notype and immunosuppressive function dif-
between CY-MDSC and TU-MDSC and provided
were two-sided.
fered. CY-MDSC consisted of higher percentage of
useful information for further strategies elabo-
Results: We showed that TLR3 expression is asso-
) and
rating the optimal immunotherapeutic protocols
ciated with superior survival in 172 HCC patients.
lower percentage of polymorphonuclear-like MDSC
based on the attenuation of immunosuppression by
TLR3 activation induced cell death in a TLR3+
means of MDSC modulation, for example by utili-
HCC cell line and promoted NK cell activation and
zation of 5-AZA or IL-12.
cytotoxicity in vitro. Injection of poly(I:C) in the
+
+
+
-
monocyte-like subpopulation (Ly6G Ly6C
+
subpopulation (Ly6G Ly6C
High
Low
) when compared
with TU-MDSC. This was accompanied by lower
+
+
+
relative expression of selected immunosuppressive
genes and lower suppression of T-cell proliferation.
After IFNγ stimulation, the expression of immunosuppressive genes increased, but the suppressive
242
1
spontaneous tumor mouse model induced 1) intraThis work was supported by grant from the Czech Science
Foundation No. P301/11/P220 and, in part, by project No.
AV0Z50520514 awarded by the Academy of Sciences of the
Czech Republic.
tumoral expression of chemokines Ccl5 and Cxcl9;
2) increased NK cell activation/infiltration into the
tumor; 3) enhanced proliferation of tumor-infiltrat243
189 | Tumor Biology and Interaction with the Immune System
190 | Tumor Biology and Interaction with the Immune System
A novel mitochondria-targeted antioxidant SkQ1: immunoregulatory properties in pancreatic cancer
B7-H1 in chemo/immune therapy of pancreatic cancer
1
2, 3
1
1
1
Yuhui Yang , Pavel P. Philippov , Jens Werner , Alexandr V. Bazhin , Svetlana Karakhanova
Alexandr V. Bazhin, Robert Ose, Jens Werner, Svetlana Karakhanova
1
Department of General Surgery, University Hospital Heidelberg, 69120 Heidelberg, Germany
Department of General Surgery, University of Heidelberg, Germany;
2
Department of Cell Signalling, Belozersky Institute of Physico-Chemical Biology and
3
Institute of Mitoengineering, Lomonosov Moscow State University, Moscow 119991, Russia
244
Reactive oxygen species (ROS) is a group of highly
ment also increased the percentage of pDCs while
Pancreatic carcinoma is one of the most aggres-
gering of specific signalling events. The inhibitory
reactive molecules containing oxygen and original-
reducing the percentage of granulocytes. In ex vivo
sive human malignancies and most lethal cancers
potential of the IFN-alpha-induced B7-H1 molecule
ly considered as a harmful byproduct of cellular
murine splenocyte cultures, SkQ1 treatment result-
worldwide. Outcome of this disease could be im-
was confirmed by co-culturing of T-cells and IFN-
respiration. However, ROS also regulates various
+
ed in a higher frequency of CD4 T-cells and CD8
proved only partially, despite recent advances in
alpha treated DC, with and without blocking of
cellular functions by participating in signaling.
T-cells as well as in a lower percentage of B cells.
surgery and chemo/radio therapies. That is why
B7-H1 with specific antibodies, and measuring pro-
Disruption in the redox homeostasis leads to the
In tumor bearing mice, SkQ1-treatment inhibited
novel approaches are strongly needed to improve
liferation and cytokine production from the T-cells.
oxidative stress and damages, and subsequently to
tumor growth and suppressed metastases. Moreo-
the treatment of pancreatic cancer patients. Immu-
Using mouse orthotopic model of pancreatic carci-
various diseases including cancer. Many antican-
ver, SkQ1 pretreatment lead to the highest percent-
notherapy represents such an option. Clinical data
noma and multiple fluorescence panels for different
+
+
cer therapies employ ROS-mediated mechanisms to
age of CD8 TEM cells and TCM cells and to the lowest
show promising results for approaches combining
subpopulations of immune cell, we demonstrated
kill cancer cells. Such approaches, either through
percentage of naïve CD8 T-cells in spleen and in
+
chemotherapeutics with activating cytokines, like
that additional blocking of B7-H1 molecule during
ROS-elevation or through ROS-depletion, demon-
the tumor. At the same time, SkQ1 had no effect
for example Interferon-alpha (IFN-alpha). However,
5-FU and IFN-alpha combinatory therapy improve
strated promising effects. SkQ1 is a mitochondria-
on Tregs, NK cells and myeloid-derived suppressor
in parallel with immune stimulating effects of this
the outcome of the treatment. The increase in anti-
targeted antioxidant which is specifically accumu-
cells. SkQ1 treatment decreased the NKT-cell fre-
treatment, the presence and induction of immu-
tumor immune response was achieved through the
lated in mitochondria and has a high efficiency in
quency in spleen and in the tumor. Like in healthy
nosuppressive mechanisms should be considered
positive effect on CD8 –T-cells and negative effect
scavenging ROS. SkQ1 was shown to have some
mice, SkQ1 pretreatment significantly increased
in the course of pancreatic cancer therapy. B7-H1
on Treg subpopulation.
anticancer activities in vivo. However, the under-
the percentage and maturation state of pDCs in
(CD274, PD-L1) regulatory molecules play an im-
Taking together, our data demonstrate that, despite
lying mechanism(s) is unclear. For regulating the
spleen and in the tumor.
portant role in the controlling and modulation of
the stimulation of anti-tumor effects, IFN-alpha
functions of immune cells a delicate oxidation-an-
For human peripheral T-cells, SkQ1 treatment
immune responses. In human pancreatic carcino-
upregulates the expression of immunosuppressive
tioxidation balance is essential. Due to the relation
showed no effect on the percentage of subpopula-
ma the B7-H1 expression is upregulated and cor-
B7-H1 molecules, which could limit the effect of
between ROS and immune system, the aim of this
tions. No differences in the expression of activation
relates strongly with poor patient’s prognosis. In
immunotherapy. Thus, it is of advantage to reduce
study is to investigate whether SkQ1 processes any
markers and regulatory molecules were detected.
pancreatic tumors B7-H1 expression contributes to
undesirable site effects of B7-H1 expression on the
immunoregulatory properties in tumor-bearing
Thus, SKQ1 does not have a direct effect on T-cells.
the tumor immune evasion and tumor progression
tumor and immune cells by additional blocking of
and healthy mice.
In conclusion, our study shows that SkQ1 has po-
and, as recently demonstrated, blocking of B7-H1
B7-H1 molecules with specific antibodies.
Survival analysis showed that SkQ1 improved the
tential anticancer activities in inhibiting pancreatic
could improve anti-tumor effects in a mouse pan-
median survival of pancreatic carcinoma bearing
cancer growth and metastasis, with SkQ1 pretreat-
creatic cancer model.
mice, implying beneficial effect of SkQ1 in anti-
ment showed the most obvious inhibition. In addi-
In our study we aimed to investigate the expression
cancer treatment. Since SkQ1 showed no direct cy-
tion, we found that SkQ1 possesses certain immu-
and function of B7-H1 molecules in the context of
totoxic effect against pancreatic cancer cell lines,
noregulatory properties in vivo. The effects of SkQ1
combined IFN-alpha therapy of pancreatic cancer.
we speculated that the improved survival probably
on various immune cell types may be responsible
Using Flow cytometry, and cytological methods we
resulted from the influence of SkQ1 on the immune
for the improved survival and for suppression of
showed that B7-H1 expression is upregulated on
system. In healthy mice, SkQ1 treatment decreased
the tumor growth and metastasis. More studies are
dendritic cells (DC) and some pancreatic cancer cell
the percentage of naïve T-cells while increasing the
still needed to understand the relation between the
lines upon treatment with IFN-alpha. As defined
percentage of memory T-cells. No difference was
effect of SkQ1 on the immune system and antican-
by Western blot approach, the IFN- alpha induced
detected for Tregs, NK and NKT-cells. SkQ1 treat-
cer activities.
expression of B7-H1 is also accompanied by trig245
191 | Tumor Biology and Interaction with the Immune System
192 | Tumor Biology and Interaction with the Immune System
Effect of platinum-containing chemotherapy on tumor
­micro-environment in gynecological malignancies
HLA class I and II antigen expression in human bladder cancer
Eveline M. Dijkgraaf, Moniek Heusinkveld, Renske Goedemans, Johan W.R. Nortier,
Marij J.P. Schoenmaekers-Welters, Judith R. Kroep, Sjoerd H. van der Burg
Javier Carretero , Ana del Campo , Aurelia Gallego , Svitlana Zinchenco , Federico Garrido ,
6
Natalia Aptsiauri
Department of Clinical Oncology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA,
Leiden, Netherlands
Departamento de Bioquímica y Biología Molecular III e Inmunologia , Universidad de Granada,
2,4,5,6
Departamento de Análisis Clínicos, Hospital Universitario Virgen de las Nieves, Granada
Spain.
1
2
3
4
5
1,5
3
Departamento de Bioquímica, Hospital Universitario Virgen Macarena, Sevilla , Spain
Monocytes attracted by tumor-induced chronic
effect is PGE2- and IL-6 dependent as blocking of
Tumor immune escape plays a critical role in cancer
inflammation differentiate to antigen-presenting
PGE2 by indomethacin and IL-6 via blocking of the
progression, but the mechanisms involved in this
cells (APC), the type of which depends on cues in
IL6-receptor using the clinically used antibody to-
process have still to be defined. In recent years, dif-
the local tumor milieu. Previously, we and others
cilizumab, restored APC phenotype and function.
ferent HLA class I abnormalities have been found in
showed the influence of cervical cancer cells and
Furthermore, STAT3 phosphorylation in APC was
tumors. The lack or downregulation of the expres-
ovarian cancer cells on APC differentiation; the
IL-6 dependent whereas pSTAT1 and 6 were PGE2-
sion of single or multiple components of HLA class
majority of cancer cells either hampered monocyte
dependent. This indicates that chemotherapy-treat-
I antigen processing pathway may help tumor cells
to dendritic cell differentiation or skewed their dif-
ed patients might benefit from concomitant therapy
to avoid the recognition and elimination by tumor-
ferentiation towards immune suppresive M2-like
with COX inhibitors and blocking of IL-6(R).
specific CD8
+
expression of HLA class I antigens was previously
prostaglandin-2 (PGE2) and interleukin-6 (IL-6). In-
reported in bladder carcinoma (Maleno et al 2006,
terestingly, enhanced COX2/PGE2 and high levels
2011) describing high frequency of the loss of het-
of IL-6 are associated with chemoresistance and
erozygosity (LOH) in chromosomes 6p21.3 (HLA
tumor progression. The influence of chemotherapy
class I heavy chain genes) (35%) and 15q21 (b2m
on the tumor microenvironment, and visa verse, is
gene) (44%).
yet unclear. Here, we studied the effects of cisplatin
We analyzed the HLA class I expression in seven
and carboplatin on APC differentiation in cervical
human bladder cell lines by flow cytometry with a
and ovarian cancer in vitro.
panel of monoclonal antibodies against HLA class
isolated monocytes were cultured in the
I and II proteins. None of the cells lines showed a
presence of tumor supernatant of cervical and
total loss of HLA class I molecules, however several
ovarian cancer cell lines treated with representa-
cases of locus-A, -B or -C downregulation were de-
tive doses of cisplatin or carboplatin that penetrates
tected. None of the studied cell lines expressed
the tumor. Strikingly, dendritic cells displayed a
HLA class II in basal condition. The HLA-class I
better survival upon chemotherapy, while M2-like
genomic typing was performed by PCR-SSO and
macrophages were most sensitive to chemothera-
the presence of LOH in chromosome 6 and 15 was
py. Treatment of PGE2 and IL-6 producing cancer
analyzed by microsatellite analysis. Preliminary
cells resulted in an increased production of these
results suggest that the studied cell lines have high
inflammatory mediators and subsequently in in-
incidence of LOH in chromosome 6 and 15, compa-
creased numbers of M2-like macrophages with
rable with the data in bladder carcinoma tissues.
CD14
Maleno I, Aptsiauri N, Cabrera T, Gallego A, Paschen A,
Lopez Nevot MA, Garrido F: Frequent loss of heterozygozity in the beta2 microglobuline region of chromosome 15 in
primary human tumors. Immunogenetics 63,65,2011
cytotoxic T lymphocytes. Altered
macrophages, depending on their ability to produce
+
Maleno I, Romero JM, Cabrera T, Paco L, Aptsiauri N,
Cozar JM, Tallada M, Lopez Nevot MA, Garrido F: LOH
at 6p21.3 region and HLA class I altered phenotypes in
bladder carcinomas. Immunogenetics 58,503,2006
increased STAT3 phosphorylation and decreased
STAT1 and STAT6 phosphorylation. This negative
246
247
193 | Tumor Biology and Interaction with the Immune System
194 | Tumor Biology and Interaction with the Immune System
Epigenetic mechanisms underlying IFNγ-induced upregulation
of antigen presenting machinery genes in tumor cells
Hyperthermic intraperitoneal chemotherapy in patients with
peritoneal carcinomatosis: Role of heat shock proteins and
­dissecting effects of hyperthermia
Veronika Polláková, Veronika Hrušková, Jana Šímová, Jana Bieblová, Marie Indrová and
Milan Reiniš
Maria Lazariotou , Malte Vetterlein , Tanja Grimmig , Christoph Thomas Germer ,
2
1
2
Joerg Pelz , Ana Maria Waaga-Gasser , Martin Gasser
Department of Tumour Immunology, Institute of Molecular Genetics, Academy of Sciences of the
Czech Republic, v. v. i., Prague
1
Department of Surgery I, Molecular Oncology and Immunology, University of Wuerzburg, Germany
2
Department of Surgery I, University of Wuerzburg, Germany
1
1
Epigenetic changes play important roles as genetic
line TC-1/A9 and RVP3 in control and DAC/TSA-
In patients with isolated peritoneal carcinomato-
alterations in carcinogenesis and in the course of
treated samples and MHC class I-deficienT-cell line
sis (PC) of gastrointestinal and ovarian cancer hy-
tumour growth. From the immunological point of
TC-1/A9 and Tramp-C2 in control and INFγ-treated
perthermic intraperitoneal chemotherapy (HIPEC)
view, it is noteworthy that downregulation of genes
samples. We have compared epigenetic agent and
represents a promising treatment option integrated
crucial for antigen presentation and costimulation
IFNγ impacts in various cell lines and identified
into multimodal concepts.
in tumour cells involve epigenetic events. It is
genes regulated by both IFNγ and the drugs. The
A retrospective study of 63 patients treated with
known that IFNγ-mediated MHC class I surface ex-
results show that although there was a clear differ-
HIPEC including a subgroup protein and gene ex-
pression is associated with upregulation of antigen-
ence in the responsiveness of the cell lines to the
pression analysis of eight subjects with different
presenting machinery genes (TAP1, TAP2, LMP2,
same treatment and, on the other hand, different
adenocarcinomas, additional PC, and from whom
LMP7). Using MHC class I-deficient tumours, we
responses of the same cells to IFNγ and epigenetic
tumors were available before and after HIPEC
have shown previously that expression of silenced
drug treatment, a set of genes (e.g. APM genes)
therapy was conducted. Relevant heat shock pro-
antigen-presenting machinery (APM) genes can
was regulated in the same manner. DNA demeth-
teins (HSPs) that may confer increased resistance
be restored by epigenetic agents (e.g. DNA meth-
ylation of selected APM genes was determined by
in tumor cells exposed to physical stress of hy-
yltransferase and histone deacetylase inhibitors).
the Methylation-Specific PCR (MSP) analysis of
perthermia were analyzed. HIPEC was performed
This work is focused on epigenetic mechanisms
different experimental and control tumour cell
under hyperthermic conditions and current chemo-
(DNA methylation, histone acetylation) in IFNγ
lines. Treatment with IFNγ, with the combina-
therapeutic protocols after cytoreductive surgery.
pathways. The principal objective was to uncover
tion of IFNγ and epigenetic agents, and with TNFα
In addition, HT-29 colon cancer cells were exposed
the role of DNA methylation in IFNγ-induced up-
induced demethylation of the promoter region of
to different hyperthermic conditions and analyzed
regulation of MHC class I, APM and costimulatory
APM genes. Our next objective was to see whether
for HSP-expression, apoptosis, and proliferation.
molecules and interferon regulatory factors (IRF)
the IFNγ signalling pathway components (IRF or
Upregulation of HSP27, HSP70/72, and HSP90 ex-
in tumour cells. Further, we have investigated
STAT1) could be influenced by 5AC and to monitor
pression was found in all tumor entities of the pa-
whether IFNγ signalling pathway components (IRF
expression of IRF genes in the TC1/A9 tumour cell
tients after HIPEC therapy (HSP90, p=0.001). An
or STAT1) could be influenced by 5-azacytidine
line after stimulation with 5AC and with IFNγ. Our
upregulation of HSP expression was also observed
(5AC). Our effort was to determine and compare
results indicate that stimulation with both 5AC and
in HT-29 cells confirming clinical results and un-
the transcription levels of selected immunoac-
IFNγ caused increased expression of these selected
derlining its dependency on preselected tempera-
tive genes and the epigenetic changes within the
IFNγ signalling pathway components. Our current
ture for optimal results as exemplified in cell vi-
genome of tumour cells after treatment with epi-
hy pothesis is that DNA demethylation of the reg-
ability. Interestingly, the optimal temperature to
genetic agents and with IFNγ. It was important to
ulatory sequences of APM genes is an important
achieve optimal toxicity on the tumor cells was
uncover whether IFNγ would act as an epigenetic
mechanism underlying enhanced expression of
found at 41°C. Therapeutic approaches like HIPEC
agent upregulating the expression of genes impor-
these genes after treatment with IFNγ.
to achieve antiproliferative and apoptosis inducing
tant for antigen presentation and costimulation
through demethylation. The aim was to describe
in detail the reversible mechanisms in tumour cell
escape from specific immunity.
We have performed a pilot study based on transcriptome analysis of MHC class I-deficienT-cell
248
1
2
cellular effects in patients with PC are negatively
This work was supported by grant from the Czech Science
Foundation No. 301/10/2174 and in part by project No.
AV0Z50520514 awarded by the Academy of Sciences of the
Czech Republic.
influenced by highly conserved HSP mechanisms
V.P. is a Ph.D. student supported in part by the Faculty of
Science, Charles University, Prague.
essary to be established to achieve optimal cyto-
in the tumor cells. This study shows for the first
time that specific hyperthermic conditions are nectoxic effects on tumor cells in HIPEC therapy.
249
195 | Tumor Biology and Interaction with the Immune System
196 | Tumor Biology and Interaction with the Immune System
Clinical significance and therapeutic potential of programmed
death 1 and programmed death ligand 1 and 2 expression in
human colorectal cancer
Correlation of tumor-associated antigens MAGE, NY-ESO-1 and
P53 expression with clinical and pathological relationships of
patients with oral squamous cell carcinoma
1
2
1
2
Martin Gasser , Maria Lazariotou , Christoph Thomas Germer , Ana Maria Waaga-Gasser
1
Department of Surgery I, University of Wuerzburg, Germany
2
Department of Surgery I, Molecular Oncology and Immunology, University of Wuerzburg, Germany
1
2
3
1
Service of Head and Neck surgery, University Hospital, Lausanne, Switzerland
2
Institute of Pathology, University Hospital (CHUV), Lausanne, Switzerland
3
Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (CHUV), Switzerland
The negative regulatory programmed death-1/pro-
the PD-L1 and PD-L2 status may be a new predictor
Despite advances in the diagnosis and treatment
MAGE-A was expressed in 52% of patients. NY-
grammed death ligand (PD-1/PD-L) pathway in
of prognosis for patients with CRC.
of Head and Neck cancer, survival rates have not
ESO-1 and P53 expression was found in 7% and
T-cell activation has been suggested to play an im-
improved over recent years. New therapeutic strate-
52% cases respectively. A higher tumor depth was
portant role in tumor evasion from host immunity.
gies, including immunotherapy, are the subject of
significantly correlated with expression of MAGE-A
Levels of immune cells expressing PD-1 in clinical
extensive research. In several types of tumors, the
proteins (p=0.03). No significant correlation could
colorectal cancer (CRC) have not been evaluated
presence of tumor infiltrating lymphocytes (TILs),
be made between the expression of both p53 and
so far. Thus PD-1 expression of tumor infiltrating
notably CD8 T-cells and dendritic cells, has been
NY-OESO-1 and histopathological parameters. Ex-
T-cells together with tumor cell derived expression
correlated with improved prognosis. Moreover, some
pression of tumor-associated antigen did not seem
of PD-L1 and PD-L2 was investigated for its clinical
T-cells among TILs have been shown to kill tumor
to impact significantly on patient prognosis.
significance in patients with CRC.
cells in vitro upon recognition of tumor-associated
As does the demonstration of p53 function inhibi-
Tumors from 116 patients with CRC (01/2001-
antigens. Tumor associated antigens are expressed
tion by CT antigens of MAGE family, our results
12/2004) were analyzed for their PD-L1 and PD-L2
in a significant proportion of squamous cell carci-
suggest, that tumor associated antigens may be
gene expression by RT-qPCR and were addition-
noma of the Head and Neck and apparently may
implicated in tumor progression mechanisms, This
ally immunostained. Outcome analyses were per-
play a role, in the regulation of cancer cell growth
hypothesis need further investigation to clarify the
formed based on completed 5 year tumor registry
notably by inhibition of p53 protein function in some
relationship between host immune response and
data.
cancers. The MAGE family CT antigens could there-
local tumor biology.
T-cell infiltration was observed in 105 (90.5%)
fore potentially be used as defined targets for im-
patient tumors with 93 (80.2%) tumors showing
munotherapy and their study bring new insight in
PD-1+ T-cells. Interestingly, PD-L expression was
tumor growth regulation mechanisms.
inversely correlated with tumor-infiltrating T lym-
Between 1995-2005 54 patients were treated sur-
+
250
1
Jean-Paul Rivals , Kishore Sandu , Snezana Andrejevic-Blant , Donata Rimoldi ,
3
3
1
1
1
Daniel Speiser , Pedro Romero , Philippe Monnier , Christian Simon and Luc Bron
+
phocytes, particularly with CD8 T-cells. Moreover,
gically in our institution for squamous cell carci-
intratumoral PD-1+ T-cell infiltration was associ-
noma of the oral cavity. Patient and clinical data
ated with advanced tumor stage (p=0.002). Pa-
was obtained from patient files and collected into
tients with PD-1+ T-cells demonstrated to express
a computerized database. For each patient, paraf-
significantly more PD-L1 on their tumor cells. In
fin embedded tumor specimens were retrieved and
addition, multivariate analysis demonstrated that
expression of MAGE CT antigens, p53, NY-ESO-1
patients positive for PD-L in their tumors and those
were analysed by immunohistochemistry Results
with PD-1+ T-cell infiltrates had a significantly
were then correlated with histopathological param-
poorer prognosis than negative patients. These data
eter such as tumor depth, front invasion according
suggest that interactions of T-cells expressing PD-1
to Bryne and both, local control and disease free
and PD-L can promote cancer progression. Thus,
survival.
251
197 | Tumor Biology and Interaction with the Immune System
198 | Tumor Biology and Interaction with the Immune System
It takes two to tango: MHC-I and Invariant chain in harmony
Endothelial cells derived from non-malignant tissues as models
for tumor vasculature are of limited value
1
1
1
3
1
Sébastien Wälchli , Shraddha Kumari , Weiwen Yang , Kine M. K. Sand , Lars-Egil Fallang ,
3
3
1, 2
3
Ole B. Landsverk , Oddmund Bakke , Johanna Olweus , and Tone F. Gregers
1
Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radium­
hospitalet, N-0310 Oslo, Norway
2
Centre for Immune Regulation, University of Oslo, N-0316 Oslo, Norway
3
Department of Molecular Biosciences, University of Oslo, N-0316 Oslo, Norway
1
2
1
1
Department of Neurosurgery, Division of Neurosurgical Research, University of Heidelberg,
Germany
2
Translational Immunology Unit, German Cancer Research Center, Heidelberg, Germany
Supplying blood vessels are not only essential for
firming endothelial identity and excluding contam-
nourishing growing tumor masses but also for fa-
ination with tumor cells or leukocytes. Unsuper-
cilitating access to invading immune cells. As a con-
vised clustering of the mRNA expression profiles
sequence tumor-derived endothelial cells became a
unveiled distinct grouping. In addition, student’s
focus in tumor immunology. The isolation and cul-
t-test showed 780 genes to be significantly differen-
Exogenous peptides are presented by specialized
similar efficiencies. Taken together, these data
tivation of tumor endothelia however are limited by
tially expressed at a p-value of 0.001 excluding low
antigen presenting cells (APC) via MHC class II
confirm the interaction between MHC-I and Ii and
the availability of freshly operated tumor material
row intensities. Following combined pathway and
molecules (MCH-II). The type II transmembrane
further demonstrate that the CLIP-replaced-Ii con-
and laborious isolation procedures. Thus model en-
gene ontology analysis identified profound enrich-
protein invariant chain (Ii) acts as a chaperone for
struct can be exploited as an efficient, proteasome-
dothelial cells derived from non-malignant tissues
ment for genes related to cell adhesion, angiogene-
MHC-II and targets it to the endosomal pathway
independent tool for peptide loading of MHC-I, with
are commonly used to imitate tumor vasculature.
sis and leukocyte migration. On basis of our mRNA
where peptide loading occurs. In addition, Ii pre-
potential application in vaccination strategies.
E.g. Human umbilical cord vein endothelial cells
profiling, we quantified two key adhesion mol-
vents the premature loading of MHC II through its
(HUVECs) are easy accessible, cheap and hence a
ecules for leukocyte transmigration, ICAM-1 (in-
CLIP region which occupies the MHC II peptide
popular model for studying angiogenesis and trans-
tercellular cell adhesion molecule-1) and VCAM-1
binding groove. Unlike MHC-II, MHC-I is expressed
migration of lymphocytes. Nevertheless, current
(vascular cell adhesion molecule-1), on the protein
in all nucleated cells and binds mainly endogenous-
publications revealed that tumor-derived endothe-
level. About one third of GECs expressed ICAM-1
ly derived peptides generated by the proteasome
lial cells harbor more alterations, through reasons
and VCAM-1 protein similar to original tumor
and targeted to the ER without the assistance of Ii.
such as vascular mimicry, than initially assumed.
tissue whereas HUVECs were negative. Cultivation
However, evidence supports that MHC I is also in-
We therefore aimed to comprehensively compare
of HUVECs in cytokine-reduced medium restored
teracting with Ii. Since the replacement of the CLIP
HUVECs to freshly isolated endothelial cells from
CAM expression. Because CAMs can be regulated
peptide has been shown to efficiently load MHC-II
glioblastoma (GECs, glioblastoma-derived endothe-
by tumor-derived factors, we moreover investigated
and to increase presentation of specific epitopes,
lial cells), a highly vascularized and aggressive
the influence of five angiogenic cytokines, proven
we wanted to determine if this held true for MHC-I
brain tumor.
by ELISA to be the most prominent ones in glioblas-
as well. We here present data showing that these
HUVECs and GECs were cultivated and harvested
toma, on CAM expression. HUVECs downregulated
proteins co-localized in the endosomal pathway
in passage one or two to minimize cultivation ar-
CAM expression after a 72 h incubation with bFGF,
when co-expressed in model cell lines. Biochemi-
tifacts. Purity of GEC isolates was confirmed by
VEGF, HGF, TGF-β1 and TGF-β2. Yet, GECs were
cal evidence suggested that MHC-I interacted with
characteristic uptake of acetylated low density li-
only sensitive to TGF-βs resulting in a 58% reduc-
Iiwt, and that this interaction was increased when
poprotein (AcLDL) and immunostainings. HUVEC
tion of ICAM-1 and 92% of VCAM-1, respectively.
CLIP peptide was replaced by known MHC-I pep-
(n=2) and GEC (n=5) expression profiles were de-
We conclude that glioblastoma-isolated endothelia
tides, indicating that this interaction was a least in
termined by mRNA microarray analysis. An assort-
show significant differences to blood vessel cells
part affected by the CLIP peptide. Next, we tested
ment of regulated genes was validated via quan-
from non-malignant tissue regarding gene expres-
the ability of the CLIP-replaced Ii to load MHC-I,
titative PCR. Subsequently, immunostainings and
sion patterns and response to external stimuli.
and showed that CD8 T-cells could indeed be ac-
flow cytometric analysis were performed to further
Consequently, if non-tumor derived endothelial
tivated. Finally, we demonstrated that dendritic
delineate functional differences.
cells are used to conclude on their behavior in
cells transfected with constructs encoding either
Nearly hundred percent of cultivated GEC showed
cancers, the suitability and cultivation method of
full length protein or the modified Ii-chain indeed
AcLDL uptake and were completely negative for
these model cells needs to be accurately proven.
primed antigen-specific naïve CD8 T-cells with
glial fibrillary acidic protein (GFAP) and CD45, con-
+
252
1
Jennifer Lohr , Andreas Mock , Philipp Beckhove , Christel Herold-Mende
253
199 | Tumor Biology and Interaction with the Immune System
200 | Tumor Biology and Interaction with the Immune System
Cytotoxic dendritic cells inhibit regulatory T lymphocyte
­generation
Helicobacter-induced preneoplastic gastric immunopathology is
suppressed by TLR2-activated B cell induced T regulatory-1 cells
Nona Janikashvili, Alexandrine Gautheron, Malika Trad, Marion Ciudad, Bernard Bonnotte
Ayca Sayi Yazgan
INSERM UMR1098, IFR 100, Faculty of Medicine, University of Burgundy, Dijon, France
1
Institute of Molecular Cancer Research, University of Zürich, Winterthurerstr. 190, Zürich,
Switzerland
2
Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical Universtiy, Maslak, Istanbul, Turkey
1, 2
1
1
, Esther Kohler , Anne Müller
Known for years as professional antigen presenting cells, dendritic cells (DC) are also endowed
with tumor cytotoxic activity. We showed that this
novel function of cytotoxic DC, together with their
ability to activate tumor specific T-cell responses,
have important implications in antitumor immu-
B-cells regulate autoimmune pathologies and
nopathology, promoting gastric mucosal homeosta-
notherapy in experimental models and in human
chronic inflammatory conditions such as autoim-
sis on the one hand and facilitating Helicobacter
ex vivo. However, cancer-induced immunosuppres-
mune encephalomyelitis and inflammatory bowel
persistence on the other.
sion often reduces the anti-tumor effects of immu-
disease. The potential counter-regulatory role of
notherapies leading to disappointing results using
B-cells in balancing pathogen-specific immune re-
such protocols in clinical trials. Therefore, thera-
sponses and excessive immunopathology is much
peutic strategies combining the induction of effec-
less understood due to the lack of appropriate per-
tive antitumor immunity with the inhibition of the
sistent infection models. We show here that B-cells
mechanisms of tumor induced immunosuppression
have the ability to negatively regulate adaptive
represent a key objective in cancer immunothera-
immune responses to bacterial pathogens. Using
py. We herein study the interactions between cyto-
mouse models of infection with Helicobacter felis, a
toxic DC and Treg the powerful suppressive cells
close relative of the human gastrointestinal patho-
responsible of the establishment and persistence of
gen H. pylori, we found that B-cells activated by
cancer-induced immunosuppression by impeding
Helicobacter TLR-2 ligands induce IL-10-producing
DC and effector T lymphocytes. We demonstrate
CD4 CD25 T regulatory-1 (Tr-1)-like cells in vitro
that cytotoxic DC generated from cancer patients
and in vivo. Tr-1 conversion depends on TCR signal-
or healthy donors resist Treg induced immunosup-
ling and a direct T-/B-interaction through CD40/
pression. Additionally, after killing tumor cells,
CD40L and CD80/CD28. B-cell-induced Tr-1 cells
cytotoxic DC inhibit the conversion of naïve T-cells
acquire suppressive activity in vitro and suppress
to Treg and, in turn, deviate their differentiation
excessive gastric Helicobacter-associated immuno-
towards the effector T helper 1 population. These
pathology in vivo. Adoptive co-transfer of MyD88-
observations emphasize important new perspec-
proficient B-cells and Tr-1 cells restores a normal
tives for the use of cytotoxic DC in cancer immu-
gastric mucosal architecture in MyD88 and ­IL-10
notherapy strategies.
mice in a manner that depends on T-cellular, but
+
+
-/-
-/-
not B-cellular IL-10 production. Our findings describe a novel mechanism of B-cell dependent Tr-1
cell generation and function in a clinically relevant
disease model. In conclusion, we demonstrate here
that the B-cell/Tr-1 cell axis is essential for balancing the control of Helicobacter infection with the
prevention of excessive Th1-driven gastric immu254
255
201 | Tumor Biology and Interaction with the Immune System
202 | Tumor Biology and Interaction with the Immune System
Investigation and inhibition of tumor immune escape from
­NKG2D-dependent NK cell cytotoxicity
Impaired expression of TAP-2 as posttranscriptionally ­controlled
by microRNAs, modulate immune escape ­mechanisms in
­esophageal adenocarcinoma
*1
*1
2
3
1
1
1, 2
1, 2
Ariane Groth , Sandra Weil , Alexander Steinle , Ulrike Köhl and Joachim Koch .
Luigi Mari , Francesca Milano , K. K. Krishnadath
1
Georg-Speyer-Haus, Frankfurt, Germany
1
2
Paediatric Haematology and Oncology Laboratory for Stem Cell Transplantation and Immunotherapy, Frankfurt, Germany
Dept. of Experimental and Molecular Medicine, Academic Medical Center, Amsterdam,
Netherlands
2
Dept. of Gastroenterology and Hepatology,Academic Medical Center, Amsterdam, Netherlands
3
Institute of Molecular Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany
*
equal contribution
+
Background: NKG2D
natural killer (NK) cells
was generated and analyzed by peptide spot arrays,
Down-regulation of the MHC class I surface anti-
one of these four microRNA binds to TAP-2 and
display cytotoxicity towards tumor cells after
flow cytometry, IP and ELISA.
gens is a mechanism of tumor cells to escape from
can inhibit transcription of this protein. Further
binding to MHC class I chain-related gene A or B
Results: We identified an sMICA-dependent tumor
immune surveillance and is common in several ag-
understanding of microRNA mediated post-tran-
(MICA/B) or the UL-16 binding proteins (ULBP)
immune escape from NK cells in NB Patients.
gressive cancers, including Esophageal adenocar-
scriptional modifications in the APM pathway can
1-5 on the targeT-cell. However, soluble NKG2D-
Within an ongoing phase I/II clinical trial with
cinoma (EAC). In a previous study we discovered
lead to the discovery of novel targets to circumvent
ligands (sNKG2DLs), which can be shed from the
IL-2-stimulated allogeneic NK (dNK) cells for im-
that in the EAC cell line OE19, downregulation of
APM-related immune escape mechanisms in solid
plasma membrane of tumor cells, might compro-
munotherapy of patients with high-risk stage IV
TAP-2, a member of the Antigen Processing and
tumors.
mise NKG2D-dependent NK cell cytotoxicity and
neuroblastoma (NB), we found high plasma levels
Presentation Machinery (APM), accounts for a de-
thus promote tumor escape from immunosurveil-
of sMICA leading to an impaired lytic activity of
ficient Dendritic Cell (DC)-mediated anti-tumor
lance. In head and neck squamous cell carcinoma
NK cells (Kloess et al., J Immunol, 2010). Further-
T-cell response. IFN-γ treatment restored TAP-2
(HNSCC) and neuroblastoma (NB) patients, elevat-
more, we were able to establish a screening system
epxression in OE19 and restored the sensitivity of
ed levels of sMICA in patient serum correlated sig-
based on spheroids as an inducible in vitro shed-
these cells to T-cell induced cytotoxicity. In OE33,
nificantly with diminished NK cell cytotoxicity. In
ding model of NKG2DLs. The hybridoma screen
another EAC cell line, TAP-2 is proficient and this
the current study we aim to elucidate the molecular
gave rise to three antibody epitope categories: one
cell line is sentitive to T-cell induced cytotoxicity.
details of this immune escape mechanism for pro-
MICA-specific and two MICA/B-specific groups.
However, silencing TAP-2 in OE33 proved to sig-
spective therapeutic intervention.
Initial depletion experiments showed that sMICA
nificantly decrease T-cell induced cytotoxicity. Un-
Methods: Since clinical results suggest a major role
and recMICA are captured by monoclonal antibod-
derstanding the molecular mechanisms at the base
of sNKG2DLs in inhibiting NK cell surveillance of
ies as well as by recNKG2D.
of these impairments can lead to circumvention of
tumor cells, NB and HNSCC tumor-cell lines were
Conclusions: Our spheroid model is a valuable tool
immune escape mechanisms and in turn the design
used to generate a 3D tumor spheroid model to
to specifically analyze NKG2D ligand shedding
of more effective treatments. Since microRNAs
study the molecular details of NKG2D-dependent
to determine the precise parameters for NKG2D-
are known as pivotal post-transcriptional regula-
tumor immune escape in vitro. NKG2DL expression
dependent immune escape from NK cells and to
tors and players in the regulation of cancer and
was verified by flow cytometry and IHC of spheroid
develop clinical intervention strategies. Therapeu-
the immune system, we analyzed the expression
cryosections. MICA shedding was induced by dif-
tic depletion of sMICA from patient serum prior
pattern of microRNAs in the above mentioned cell
ferent stress conditions. Additionally, infiltration
to cell-based therapies could significantly boost
lines and found significant differential expression
and susceptibility to primary NK cell cytotoxicity
donor NK cell cytotoxicity and improve the clinical
of several microRNAs. More specifically we found
was analyzed. In parallel, a therapeutic interven-
benefit for NB patients.
that treatment of OE19 with IFN-γ induced down-
tion approach to deplete sMICA from NB patients
`sera was initiated. To functionalize a suitable bio-
256
regulation of four microRNAs, which were also
[email protected]
expressed at very low levels in OE33. By analys-
reactive surface, a panel of monoclonal antibodies,
ing databases for the predicted microRNA targets
raised against the alpha3-helical domain of MICA,
and target down-regulation scores, we found that
257
203 | Tumor Biology and Interaction with the Immune System
204 | Tumor Biology and Interaction with the Immune System
Expression and regulation of arginine transport proteins
in ­human T lymphocytes
Arginine auxotrophy: tumor growth analysis in a 3D in vitro
culture system
1, 2
2
1
2
Vanessa Schnitzius , Alice Habermeier , Claudia Luckner-Minden , Jean-Paul Boissel ,
3
3
2,*
1,*
Mario Hubo , Helmut Jonuleit , Ellen Closs , Markus Munder
Jose Hadi Sutanto, Katharina Schneider, Claudia Luckner-Minden, Hakim Echchannaoui,
Edite Antunes, Matthias Theobald, Markus Munder
1
Third Department of Medicine (Hematology, Oncology, and Pneumology), University Medical
Center of the Johannes Gutenberg University Mainz, Germany
Third Department of Medicine (Hematology, Oncology, and Pneumology), University Medical
Center of the Johannes Gutenberg University Mainz, Germany
2
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Germany
3
Department of Dermatology, University Medical Center of the Johannes Gutenberg University
Mainz, Germany
*
these authors share last authorship
The adaptive anti-tumor immune response is inhib-
CAT-1 mRNA levels were preserved specifically in
Metabolic profiling of tumor cells offers the unique
vivo reality we then analysed arginine-, citrulline-
ited by myeloid-derived suppressor cells (MDSC),
the absence of arginine while in the presence of ar-
potential to specifically target malignancy based
and ASS-dependent tumor cell growth in a three-
which expand in tumor patients systemically and
ginine hCAT-1 mRNA decreased again. In contrast,
on the identification of auxotrophy for specific nu-
dimensional (3D) tumor model. We established mul-
locally within the tumor microenvironment. MDSC
CAT-3 mRNA (previously thought to be central
trients. Depletion of the amino acid arginine is an
ticellular tumor spheroids (MCTS) of immortalised
mainly suppress T-cell responses via arginase-medi-
nervous system specific) was expressed in resting
effective treatment strategy for tumors that are de-
murine embryonic fibroblasts (MEF) with different
ated arginine depletion. Since arginine availability
human T-cells and preserved upon stimulation in the
ficient in the enzyme argininosuccinate synthase
(low, medium, and high) expression levels of ASS
is crucial for full T-cell activation and consecutive
absence of arginine. In contrast, it was downregu-
(ASS), which converts citrulline into arginine.
and compared two-dimensional and three-dimen-
T-cell-mediated anti-tumor immune responses, we
lated upon activation in the presence of arginine. The
ASS-deficient tumors are unable to use citrulline
sional tumor cell viability and growth sequentially
have started to analyse the expression and regula-
CAT-2 mRNA isoforms were not expressed in human
from the microenvironment as a rescue strategy to
by (i) MTS tetrazolium salT-cell proliferation assay,
tion of arginine transport proteins in primary human
T-cells under any tested condition, contrasting with
bypass arginine deficiency. Arginine depletion can
(ii) microscopic MCTS diameter measurements, (iii)
T lymphocytes. Transmembranous transport of the
their prominent inducible role in activated murine
be achieved endogenously via arginase-expressing
flow cytometric annexin V / propidium iodide anal-
cationic amino acid arginine has so far been studied
myeloid cells as well as other eukaryotic tissues.
myeloid cells but also pharmacologically via ap-
ysis and (iv) flow cytometric cell division analysis
in other eukaryotic tissues and it has been shown
mRNA of the system y+L transporter y+LAT2 was
plication of pegylated forms of arginine-metabolis-
by eFluor® 670 labeling. We show that MEF growth
that it can be shuttled across cell membranes via spe-
strongly expressed in resting human T-cells and
ing enzymes like arginase or arginine deiminase,
is critically dependent on arginine and that citrul-
cialised membrane proteins that only transport cati-
further induced upon activation. The elvated expres-
already tested in clinical phase I-III studies. Since
line can rescue MEF proliferation only if ASS is ex-
onic amino acids (CATs, cationic amino acid trans-
sion was preserved in the absence, but not the pres-
human T lymphocytes can upregulate ASS upon
pressed. In contrast to 2D culture conditions, MEF
porters) or via transporter proteins for cationic and
ence of arginine at 48h. CAT-1 and CAT-3 protein ex-
arginine depletion and reconstitute functional
MCTS cellular viability is preserved for extended
neutral amino acids (y+LATs, b AT und ATB ).
pression paralleled the respective mRNA in human
defects upon citrulline supplementation, arginine
periods of time independent of extracellular ar-
In contrast to other tissues, arginine transport into
T-cells as shown by Western Blots. Finally, expres-
depletion with concomitant citrulline supplemen-
ginine: while MEF cell viability is decreased dra-
human immune cells is largely a black box, so far.
sion and regulation of CAT isoforms and y+LAT2
tation is a very promising treatment strategy to
matically after 2 days without arginine, 3D culture
0,+
0,+
+
+
For all our experiments we used primary human T
was similar in both CD4 and CD8 human primary
simultaneously induce cancer cell death and pre-
conditions allow preservation of viability for at least
lymphocytes that were sorted from normal blood
T-cells as well as highly purified human regulatory
serve anti-tumor immune cell functions.
6 days without exogenously substituted arginine.
Also, 3D arginine-deprived MCTS can regain normal
+
+
donors by magnetic separation. We stimulated
CD4 CD25 T-cells.
To analyse the prerequisites for this strategy we first
primary human T-cells by anti-CD3/anti-CD28-cou-
In summary, our results underscore the complexity
measured tumor cell growth ( H thymidine incor-
growth after replenishment of arginine.
pled beads for 6h, 24h, and 48h in the absence or
of immune cell regulation via arginine availability
poration) and metabolic mitochondrial activity in a
In summary, our results demonstrate the impor-
presence of arginine (1 mM) and prepared mRNA
and contribute to a better understanding of adap-
panel of various established tumor cell lines in the
tance of arginine metabolism for tumor cell growth
as well as cellular protein lysates. In parallel, we
tive immunity also in the context of tumor-associ-
absence or presence (1 mM) of arginine +/- citrul-
and underscore the need for the development of
monitored arginine-dependent T-cell functions by
ated immune escape. Arginine transport proteins of
line (1 mM) substitution and correlate these results
appropriate in vitro model systems as a first step
proliferation and cytokine secretion analysis assays.
human T lymphocytes are potential new targets for
with tumor cell expression of ASS. We show that (i)
for later therapeutic manipulation of amino acid
By quantitative Real Time PCR we found a complex
the pharmacological manipulation of T-cell functions.
all tumor cell lines need arginine for growth and
metabolism in the tumor microenvironment.
(ii) that only ASS-positive tumor cells are able to
regulation of CAT isoforms and y+LAT2 upon T-cell
258
3
activation: CAT-1 mRNA was significantly induced
Funding: DFG (MU 1547/4-1 to M.M. and CL 100/6-1 to E.C.)
use exogenously applied citrulline to compensate for
in 6h after stimulation. At later time points, high
Note: This abstract contains parts of the M.D. thesis of V.S.
arginine deficiency. In order to better recapitulate in
Note: This abstract contains parts of the M.D. theses of
J.H.S. and K.S.
259
205 | Tumor Biology and Interaction with the Immune System
206 | Tumor Biology and Interaction with the Immune System
Immunomodulatory properties of cancer stem cells isolated
from human glioblastoma and colorectal cancer
Radiation-induced gene expression leads to altered immune
signaling and migration in glioma-initiating cells
1
1
2
2
1
Cristina Maccalli , Andrea Volontè , Ena Wang , Francesco M. Marincola , Giorgio Parmiani
1
2
Unit of Immuno-biotherapy of Melanoma and Solid Tumors, San Raffaele Foundation Scientific Institute, Milan, Italy; Infectious Disease and Immunogenetics Section (IDIS)
Department of Transfusion Medicine, Clinical Center, and Center for Human Immunology
(CHI) National Institutes of Health, Bethesda, MD, USA
260
1, 2
2
3
3
Nicola Hoppmann , Christoph Schmitz-Salue , Gabriela Salinas , Lennart Opitz ,
2
2
1
Ella Kim , Alf Giese , Frauke Zipp
1
Department of Neurology, University Medical Center Mainz, Germany
2
The Translational Neurooncology Research Group, Department of Neurosurgery,
University Medical Center Mainz, Germany
3
Transcriptome Laboratory, University Medical Centre Göttingen, Germany
We have previously reported that glioblastoma
activity by blocking this cytokine with a neutral-
Glioblastoma multiforme (GBM) is the most common
Therefore, the identification of molecular mecha-
multiforme (GBM)-derived cancer stem cells (CSCs)
izing antibody leading to an efficient in vitro in-
and most aggressive tumor of the central nervous
nisms that render GICs capable of withstanding
have a low immunogenic profile, eliciting mostly
duction of TH1 type responses in the autologous
system in adults. The standard treatments for GBM,
cytotoxic treatments is pivotal also for further de-
TH2-mediated responses in autologous settings
co-culture of CSCs and PBMCs.
including surgery followed by the combined radia-
velopment of the immune-based glioma therapy.
and inhibiting allogeneic T-cell proliferation com-
Of note, we could also identify a CSC-associated
tion- and chemotherapy, only modestly improve
pared to their FBS-cultured non-CSC (FBS tumor
microRNA signature. We are currently exploiting
patient survival. Post-treatment recurrence due to
cells) pairs (Di Tomaso et al, 2010). In addition,
these results to identify microRNA with immu-
radio- and chemoresistance is the major cause of
CSCs isolated from colorectal cancer (CRC) tissues
noregulatory functions.
fatality in glioma patients. Stem-like Glioma Initiat-
are being characterized both functionally and im-
Altogether, these results will allow to identify im-
ing Cells (GICs) are thought to play a major role in
munologically; early data suggest features compa-
munomodulatory agents that can efficiently restore
generating glioma resistance to different types of
rable to those of the GBM model. A candidate nega-
the expression of immunogenic molecules on CSCs
cytotoxic treatments, but molecular mechanisms
tive immunoregulatory molecule is represented by
and, thus, to design new immunotherapy protocol
underlying the resistance remain poorly defined.
the indoleamine 2,3-dioxygenase (IDO), a molecule
for GBM and/or CRC patients.
The aim of this study was to identify molecular
involved in the generation of immune tolerance.
mechanisms involved in the acquisition of a radi-
By RT-PCR a preferential increase of the mRNA
oresistant phenotype in malignant gliomas through
of this molecule in CSCs vs FBS tumor cells was
comparative assessments of genome-wide transcrip-
detected after IFN-γ treatment. We assessed the
tional profiling of non-irradiated GICs and their in
functional activity of IDO determining, by a col-
vitro created radioresistant derivatives using Gene-
orimetric assay, IDO-mediated tryptophan catabo-
Chip Human Gene 1.0 ST arrays (Affymetrix).
lism in culture supernatants. In most (6/8) cases a
Our analysis revealed that the exposure to clini-
higher activity was associated with IFN-γ treated
cally relevant doses of ionizing radiation induces
CSCs but not with their FBS tumor counterparts.
differential expression of genes that are involved in
Interestingly, IDO-mediated activity was inhibited
immune signaling and migration/adhesion.
by treatment of these cells with the specific inhibi-
The results indicate that radiation-induced changes
tor 1-Methyl Tryptophane (1-MT). Furthermore, by
in adhesion and immune signaling molecule ex-
blocking IDO in GBM CSCs we could both recover
pression might lead to tumor progression, immune
T-cell proliferation during the co-culture with allo-
response and inflammation via altered intracellu-
geneic PBMCs from healthy donors and induce TH1
lar signaling pathways as well as cross-talk com-
type responses in autologous settings.
munication with the tumor microenvironment.
In addition, we found that CRC CSCs expressed IL-4
This eventually limits the efficacy of local therapies
and demonstrated its negative immunoregulatory
and contributes to immune evasion mechanisms.
261
207 | Tumor Biology and Interaction with the Immune System
208 | Tumor Biology and Interaction with the Immune System
Selective BRAF inhibition decreases tumor-resident lymphocyte
frequencies in a mouse model of human melanoma
Synchronous BRAF V600E and MEK inhibition leads to superior
control of melanoma by limiting MEK inhibitor induced skin
toxicity
1*
1*
1, 2, 3
Anna I. Hooijkaas , Jules Gadiot , Christian U. Blank
1*
1*
1, 2
*
These authors contributed equally to this work
Jules Gadiot , Anna I. Hooijkaas , Christian U. Blank
1
Division of Immunology and 2 Division of Medical Oncology, Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX, Amsterdam, Netherlands
*
These authors contributed equally to this work
1
Division of Immunology and 2 Division of Medical Oncology, Netherlands Cancer Institute,
Plesmanlaan 121, 1066 CX, Amsterdam, Netherlands
The development of targeted therapies and im-
-/-
The MAP Kinase and PI3 Kinase pathways have
Combining the selective BRAF and MEK-inhibi-
munotherapies has markedly advanced the treat-
BRAFV600E/PTEN
melanomas. More strikingly,
been identified as the most common pathways that
tors led to superior long-term tumor control due
ment of metastasized melanoma. Building on the
PLX4720 treatment led to a decreased frequency of
mediate oncogenic transformation in melanoma.
to reduced MEK inhibitor induced skin toxicity.
observation that immune recognition is a frequent
tumor-resident T-cells, NK-cells, MDSCs and mac-
Therefore, the majority of compounds developed
Histological analyses confirmed the skin protec-
event in melanoma, a series of immunotherapeutic
rophages, which could not be restored by the ad-
for melanoma treatment target either one of these
tive effect of BRAF inhibition towards the MEK
approaches has been evaluated in clinical trials,
dition of anti-CTLA-4 mAb. As this effect was not
pathways. Beside such targeted therapies, immu-
inhibitor induced skin toxicity. Analyses of pERK
culminating in the first phase III study that showed
observed upon treatment of BRAF-wildtype B16F10
notherapeutic approaches have shown promising
levels within the keratinocytes could not convinc-
an improved overall survival of melanoma patients
tumors, we conclude that the decreased frequency
results. A combination of targeted and/or immu-
ingly explain the protective effect of BRAF inhibi-
since twenty years. Furthermore, recent advanc-
of immune cells correlates to BRAF
inhibi-
notherapies could potentially result in further im-
tion. Analysis of tumor infiltrating T-cells, as well
es in our understanding of the genetic lesions in
tion in tumor cells and is not due to an off-target
provement of treatment outcome. In order to pre-
as gene-expression profiling of skin from BRAF
human melanoma now also allow the specific tar-
effect of PLX4720 on immune cells. Furthermore,
clinically identify efficient treatment combinations
and/or MEK inhibitor treated animals is currently
geting of the signaling pathway alterations in this
anti-CTLA-4 mAb treatment of inducible mela-
and to optimize therapy protocols in terms of e.g.
ongoing.
V600E
disease. While treatment with selective BRAF
noma mice treated with PLX4720 did not result in
sequence and timing, mouse models will be re-
Our Tyr::CreER ;PTEN ;BRAF
inhibitors (like vemurafenib or dabrafenib) leads
enhanced tumor control, while anti-CTLA-4 mAb
quired.
melanoma model indicates that MEK inhibitor
to high response rates but short response duration,
treatment did improve the effect of tumor-vaccina-
We
the
treatment can be intensified when combined with
anti-CTLA-4 blocking therapies induce sustained
tion in B16F10-inoculated mice.
Tyr::CreER ;PTEN ;BRAF
inducible mela-
BRAF inhibition. Furthermore we envision that
responses, but only in a limited number of patients.
Our data suggest that for those patients in which
noma model on a C57BL/6J background. Tumors
this model is valuable for preclinical testing of com-
The combination of these diametric treatment ap-
vemurafenib treatment does not induce cell death,
from this model harbor the BRAF
proaches may further improve survival, but pre-
this drug may negatively affect the immune activ-
are PTEN-deficient, making them highly suitable
clinical data concerning this approach is limited.
ity within the tumor. Consequently, these patients
for the testing of targeted therapies.
inhibition can
might not benefit from the addition of anti-CTLA-4
Selective inhibition of BRAF
synergize with anti-CTLA-4 monoclonal antibody
mAb treatment to selective BRAF inhibition. On a
ment of melanoma bearing mice resulted in a strong
(mAb) treatment, focussing on the interaction
more general note, the potential effects of targeted
decrease of tumor outgrowth, but tumor regression
inhibitor PLX4720 and the
therapy on the tumor-microenvironment should be
was not observed. Targeting the downstream sign-
immune system. In this study we used the immune
taken into consideration in the design of clinical
aling protein MEK by the inhibitor GSK1120212B
trials combining targeted and immunotherapy.
resulted in a stronger growth inhibition and even
V600E
We investigated whether BRAF
V600E
between the BRAF
T2
-/-
F-V600E/+
competent Tyr::CreER :PTENF ;BRAF
in-
V600E
have
T2
crossed
F-/-
and
characterized
F-V600E/+
V600E
V600E
-mutation and
T2
F-/-
F-V600E/+
inducible
binations of different targeted therapies as well as
combinations with immunotherapy.
by PLX4720 treat-
ducible melanoma mice of which all mice develop,
limited regression of larger malignancies. However,
within one month after tumor induction, a rapidly
GSK1120212B led to the development of serious
-/-
262
tumor growth, it did not induce cell death in
growing BRAFV600E/PTEN melanoma with his-
skin-toxicity comparable to the dose-limiting tox-
tology similar to human spindle cell melanoma.
icity that is observed in melanoma patients treated
While PLX4720 treatment strongly decreased
with MEK inhibitors.
263
209 | Tumor Biology and Interaction with the Immune System
210 | Tumor Biology and Interaction with the Immune System
Human skin-derived and lymph node-resident dendritic cell
Clinical implications of immune evasion in microsatellite-­
unstable colorectal cancer
subsets display differential T-cell stimulatory activity and are
differentially modulated by primary melanoma tumors and
metastases in the sentinel lymph node
1
2
2
Department of Pathology, VU University medical center, Amsterdam, Netherlands
2
Department of Medical Oncology, VU University medical center, Amsterdam, Netherlands
3
Department of Surgical Oncology, VU University medical center, Amsterdam, Netherlands
1
1
1
Department of Applied Tumour Biology, Institute of Pathology, University of Heidelberg,
­Heidelberg, Germany and Collaboration Unit Applied Tumor Biology, German Cancer Research Center (DKFZ), Heidelberg, Germany
2
Department of Immunology, University of Pittsburgh, PA, USA
Background and Aims: High level microsatellite
detected in 35%, 12%, and 2% of HLA class II an-
instability (MSI-H) is encountered in 10 to 15% of
tigen-negative MSI-H CRC lesions. No significant
all colorectal cancers (CRCs) as a result of DNA
correlation between tumor stage and HLA class II
Melanoma tumors escape immune surveillance
and B7.H4 co-inhibitory receptors. These data thus
mismatch repair deficiency. MSI-H CRC represents
antigen status was observed; however, MSI-H CRC
in early stages of their development through the
support a dominant role for LN-resident DC subsets
a valuable model for studies on tumor immunology
lesions that harbored mutations of the HLA class
release of suppressive factors that condition the
in T-cell activation.
and immune evasion, because well-defined MSI-
II-regulatory genes displayed significantly higher
primary tumor site and draining lymph nodes to
ably, lower frequencies of the skin-derived subsets
H-specific antigens directly result from mismatch
numbers of tumor-infiltrating CD4-positive lym-
tolerate and support invasion and metastasis. Most
were found in tumor positive SLN, whereas their
repair deficiency-induced mutations affecting mi-
phocytes.
notably, a reduced frequency and activation state
phenotypic activation state was unaffected by SLN
crosatellites in gene-encoding regions. We here
Conclusions: Our results suggest that immune
of dendritic cells (DC) in the initial tumor-draining
status. The reverse was the case for the LN-resi-
systematically analyzed MSI-H CRCs for the pres-
evasion caused by abrogation of HLA class I and
lymph node, the so-called sentinel lymph node
dent subsets (both conventional and plasmacytoid)
ence of immune evasion phenomena by examining
II-mediated antigen presentation represents a
(SLN), may well interfere with the activation of an-
with unaffected frequencies but reduced activation
genes coding for components of the cellular HLA
common and functionally relevant phenomenon
ti-tumor effector T-cells and thus contribute to the
state in tumor positive SLN. Of note, CD83 levels
class I and II antigen presentation machinery for
occurring during the pathogenesis of MSI-H CRC.
early metastatic events that characterize this tumor
on LC in tumor negative SLN showed a significant
structural alterations. The results were related to
The absence of distant metastases in patients with
type. We have analyzed DC subset frequencies and
reverse correlation with Breslow thickness of the
clinical and histopathological parameters.
B2M mutation-positive CRC lesions underlines
activation state by multicolor flow cytometry in
primary tumor and increased with longer time in-
Methods: Bioinformatics tools were used to iden-
the clinical significance of B2M mutations for the
SLN samples from untreated or saline-administered
tervals between excision of the primary tumor and
tify genes relevant for antigen processing and pres-
natural course of the disease. Moreover, the obser-
patients with stage I-III melanoma (n=27) who par-
the SLN. These findings indicate a dominant sup-
entation that encompassed coding microsatellites
vation of increased T-cell infiltration in tumors har-
ticipated in clinical trials on the effects of local im-
pressive effect of the primary tumor on the activa-
as mutational targets in mismatch repair-deficient
boring mutations of HLA class II-regulatory genes
munomodulation and identified and characterized
tion state of skin-derived DC subsets in SLN and
cancers. Microsatellites were identified in Beta2-
suggests that HLA class II antigen-negative tumor
two skin-derived and two LN-resident subsets (van
metastasis-related suppression of SLN-resident DC
microglobulin (B2M) and the HLA class II-regula-
cell clones may be selected for in an environment
de Ven et al. Blood 118:2502, 2011).
subsets, and are in keeping with localized inhi-
tory genes and examined for the presence of muta-
enriched for anti-tumoral immune cells. Taken to-
In a comparative study with skin-migrated DC, two
bition by the tumors of their microenvironment.
tions in a set of clinically well-characterized CRC
gether, these data highlight that abrogation of HLA-
Langer-
Moreover, they suggest that primary melanoma-
specimens.
mediated antigen presentation is very frequent in
hans Cells (LC) with intracellular Langerin and
mediated suppression of activation and migration
Results: MSI-H CRCs displayed truncating muta-
MSI-H CRC lesions. Mismatch repair deficiency
of cutaneous DC subsets enables local metastasis.
tions of the B2M gene in approximately 30% of
thus not only contributes to the generation of im-
lesions, and B2M mutations were closely correlated
munogenic frameshift peptide antigens, but also
with a lack of HLA class I antigen expression on the
to the abrogation of HLA class I- and II-mediated
as CD14-BDCA3 CD103 and CD14 BDCA3 CD103 ,
tumor cell surface. Correlation with clinical data
antigen presentation pathways.
suggesting cross-priming ability. Despite their
revealed that B2M mutation frequency increased
higher maturation state, skin-derived DC (and LC
with local tumor stage. However, B2M mutations
in particular) proved inferior in T-cell induction
were absent in distant metastasis-positive MSI-H
and accompanying IFNγ release. This might be
CRC lesions. Mutations inactivating the HLA class
related to their higher expression levels of the B7.H1
II-regulatory genes RFX5, CIITA, and RFXAP were
+
CD1a subsets were identified as CD11c
int
hi
E-Cadherin expression and as CD11c Dermal DC
with variable expression of Langerin. Two other
-
CD1a LN-residing DC subsets were characterized
hi
264
1
3
Mari F.C.M. van den Hout , Bas D. Koster , Alfons J.M. van den Eertwegh , Berbel Sluijter ,
3
3
1
3
Barbara G. Molenkamp , Sybren Meijer , Rik J. Scheper , Paul A.M. van Leeuwen , Rieneke
2
3
2
van de Ven , M. Petrousjka van den Tol , and Tanja D. de Gruijl .
1
1
Matthias Kloor , Anita Voigt , Eva-Maria Surmann , Anna Tikidzhieva , Miriam Reuschen1
1
1
2
1
bach , Kathrin Bauer , Sara Michel , Soldano Ferrone , Magnus von Knebel Doeberitz
-
+
lo
+
265
211 | Tumor Biology and Interaction with the Immune System
212 | Tumor Biology and Interaction with the Immune System
Cytokines in bone marrow and peripheral blood of breast
­cancer patients as the prognostic signs of tumor progression
Investigation the interferon-alpha as a modifier of epithelial –
mesenchymal transition of malignant cells in vitro
1
1
2
1
Yuri Kudryavets , Nadiia Semesiuk , Andrij Zhylchuk , Natalya Bezdenezhnykh ,
1
2
1
­Alexandra Lykhova , Victor Zhylchuk , Ada Vorontsova
Yuri Kudryavets, Natalya Bezdenezhnykh, Alexandra Lykhova, Nadiia Semesiuk,
Ada Vorontsova
1
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of
Ukraine, Kyiv, Ukraine
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Kyiv, Ukraine
2
Rivne Region Hospital, Rivne, Ukraine
266
Breast cancer is the most frequent type of cancer in
donors (<10 pg/ml). However, the TNF level more
The understanding of the processes of epithelial to
41,3±1,2% - in IFN-modified); has been found a
women. Despite the improvement in detection and
than 150 pg/ml (134±30pg/ml) was observed
mesenchymal transition (EMT) and mezenchymal-
very interesting fact – the appearance of E-cadherin
treatment, ~30% of newly diagnosed women with
in 28,5% of breast cancer patients in peripheral
epithelial transition (MET) to tumor progression
positive K-562 cells after long-term treatment of cy-
breast cancer will die. In most cases, death results
blood only group of progression. At the same time
and monitoring of these processes in clinical prac-
tokine. This phenomenon associated with inhibi-
from the dissemination of cancer cells through
in BM such level of TNF was observed in 57,9%
tice is very important today. Also very important
tion of biological properties malignancy behavior
lymphatic or blood vessels and the development of
(197,7±27pg/ml) patients in group of progression
is the search for modifiers of these processes, as it
of tumor cells.
distant metastases. The prognostic significance of
compared with the 19% (110,3±19pg/ml; p<0,02)
will provide new targets for treatment and help to
From these data we can conclude that IFN can
disseminated tumor cells (DTC) in the bone marrow
cases in group of remission.
change routine schemes of therapy. We think that
be involved in the control of EMT/MET programs
(BM) of breast cancer patients at present time is well
Biological activity of CSFs in samples of plasma BM
this modifier can be interferon-alpha (IFN).
of tumor cell transdifferentiation because all the
known. Yet, in many cases progression of tumor
breast cancer patients was 540±33 U/ml and don’t
In our study we have used as the experimental
studied proteins and transcriptional factors are
growth is not associated with the detection of DTC
depend on the clinical stage of disease. However
model of human tumor cells (nonsmall-cell lung
markers of these processes. So the modifying
in BM. It is not excluded that the level of particu-
the level of these factors in peripheral blood in
cancer cells of А-549 line, HeLa – cervical carci-
action of IFN on the process of EMT may be a new
lar cytokines may serve as the additional factors of
breast cancer patients group of progression was
noma, K-562 – chronic myelogenous leukemia)
mechanism of its antitumor activity.
prediction progression of primary tumor growth in
significantly increased (at 72,5%) in 60% patients
which were prolonged exposure with IFN. It was
such patients. BM aspirates from 50 patients were
in compare with that in group remission (p<0,01).
shown that the long-term exposition of A-549 cells
screened for DTC by immunocytochemistry with
It is important that the increased levels of that
to cytokine leads to reliable decrease in the number
the pancytokeratin mAb clones AE1/AE3, according
cytokines were associated with tumor recurrence
of cells expressing vimentin and leads to strong
to the ISHAGE recommendation. The levels of cy-
even in the absence of DTC in the BM.
increase in the number of cells, which express E-
tokine (tumor necrosis factor - TNF, colony stimulat-
The level of VEGF in BM plasma was significantly
cadherin (from 22±1,3% in control to 63± 2,6% in
ing factors – CSFs and interferon alpha - IFNa) and
increased in all of patients regardless of their clini-
IFN-modified). At the same time as revealed that
it’s biological activity in plasma of peripheral blood
cal status and was 295±55pg/ml and 322±56pg/ml
the long-term exposition of A-549 cells to IFN leads
and bone marrow were tested in vitro using stand-
in group of progression and remission, respectively.
to decrease N-cadherin-positive cells (from 100% in
ard cell culture methods. All of the patients were
Interesting that in a stage of disease progression
control to 46± 4,1% in IFN-modified). Importantly
divided in to two groups of clinical status: “progres-
endogenous IFNa was detected in BM of 2 patients
that there was a significant reduction in the level
sion” group (23 patients with disease progression;
(11%) only, however in patients of remission group
of expression of EMT transcription factors Twist
T1-4N0-2M0-1) and “remission” group (27 patients with
IFNa was detected in BM of 26% cases (18±2,6IU/
and Slug in cells after long-term treatment of IFN.
remission; T1-4N0-2M0). The level of VEGF in BM was
ml) and in their blood elevated levels of IFNa
The same data – increase of E-cadherin expression
tested by ELISA.
(19,4±26IU/ml) was determined in 69% of patients.
and decrease of N-cadherin and Vimentin expres-
DTC presence in samples of BM was associated
Thus, detection DTC in bone marrow and deter-
sion – we’ve got after prolonged treatment with
with stage as a rule of tumor recurrence and was
mination the level of TNF, IFN and CSFs in plasma
IFN of other tumor cells, such as: HeLa (21,4±3,1%
revealed in 50% breast cancer patients of “progres-
peripheral blood and BM of breast cancer patients
E-cadherin and 100% N-cadherin positive cells in
sion” group. The level of TNF in peripheral blood
could be very important complex of signs for prog-
control, 93± 4,1% E-cadherin and 54,2± 4,4% N-
of breast cancer patients was increased (more 60
nosis metastatic process and correction antitumor
cadherin positive cells in IFN- modified) and K-562
pg/ml) in all cases compared with that of healthy
individualized therapy.
(88±3,6% Vimentin positive cells in control and
267
213 | Tumor Biology and Interaction with the Immune System
214 | Tumor Biology and Interaction with the Immune System
Dynamic Capture of Tumor Cell Propagation with Associated
­Stromal and Immune Responses in CNS Tumor Microenvironment
Different tumor suppressors control expression of ULBP2, an
­innate stress ligand of the activating lymphocyte receptor NKG2D
Agne Petrosiute, Jay Meyers, Deborah Barkauskas, Joseph Nthale, Alex Y. Huang
Anja Heinemann , Fang Zhao , Alexander Steinle , Sven Diederichs , Dirk Schadendorf ,
1
Annette Paschen
1
1
2
3
1
Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio, U.S.A.
Ex vivo experimental systems often lack the neces-
results directly demonstrate our ability to develop an in
sary conditions to fully capture complex intercellular
vivo imaging platform for not only tumor propagation
communications between tumor cells and surround-
studies, but also real-time monitoring of immunothera-
ing tissues – a critical feature in understanding cancer
peutic strategies in intact tissues of live experimental
development and immune evasion. Common in vivo
animals.
imaging modality such as bioluminescence lacks the
In order to extend our in vivo observations to include in-
resolution necessary to discern subtle structural dif-
terrogation of CNS tumor-associated immune respons-
ferences and heterogeneity in the tumor niche, or to
es, we employed syngeneic murine tumor systems
visualize cellular activities on a single-cell level. On
in which both adaptive and innate immunity can be
the other hand, microscopic examinations of fixed
assessed in the CNS tumor microenvironment. We
specimens are devoid of the 3-dimensional context or
focused on medulloblastoma (MB), the most common
sequential evolution of tumor progression within the
malignant pediatric CNS tumors with a propensity
same host. These limitations are true not only for the
for extracranial dissemination. We examined cellular
understanding of anti-tumor immunity in the periph-
mechanisms by which MB recruits and functionally
ery, but especially true for studying anti-tumor immune
tolerize cells of the immune systems such as microglia,
responses in the central nervous system (CNS).
peri-vascular macrophages, dendritic cells and antigen-
Recent new insights have come from studies involving
specific / antigen-non-specific lymphocytes in CNS mi-
the use of intravital 2-photon laser scanning microsco-
croenvironment. Using murine MB cell lines derived
py (2P-LSM), a technique which allows deep visualiza-
from Patched /p53 mice, we assessed immune cell
tion (>300um) with single-cell resolution (<1um), thus
infiltration of intracranially implanted MB tumors by
enables direct observation of cellular behavior in intact
flow cytometry, immunohistology and 2P-LSM. Using
tissues at a suitable dynamic spatial-time resolution.
cell-specific fluorescent reporter mice such as CX3CR1-
Our laboratory studies the role of tumor microenviron-
GFP or CD11c-GFP recipient mice, we found infiltra-
ment in shaping immune repertoire and develops strat-
tion of CX3CR1 , CD11b /CCR2 , F4/80+ cells, and
-/-
+
+
+
+
+
+
egies to modify tumor microenvironment to enhance
CD4 CD25 Foxp3 T-cells in response to implanted
anti-tumor immunity. One such example is our recent
MB tumor cells. We are currently testing the effect of
collaborative study with investigators at the Cleveland
tumor-associated chemokines in orchestrating these
Clinic focused on glioblastoma multiforme (GBM),
cellular recruitments, and how perturbation of the
which is thought to contain a cellular hierarchy with
chemokine axis may affect outcome of anti-tumor im-
+
a CD133 sub-population representing self-renewing
munity.
and tumorigenic GBM stem cells (GSCs). In a xeno-
Using 2P-LSM and a CNS-inflammatory model, the
transplant model, a single GSC was capable of tumor
experimental autoimmune encephalomyelitis, we
initiation in the mouse brain. To directly test the rela-
have also begun to undercover the role of perivascular
+
tive tumorigenic potential of GSCs (CD133 ) and non-)
268
+/-
antigen-presenting cells and microglia in guiding the
GSCs (CD133 , we inoculated paired tumor populations
recruitment of CNS-bound lymphocytes. The combina-
from the same primary GBM tumor cells and monitored
tion of all of these approaches – in situ dissection of
tumor competition by serial 2P-LSM through implanted
tumor biology and immune cell recruitment – holds
cranial windows. Serial 2P-LSM imaging experiments
promise to enhance our ability to harness immune cell-
showed that after 35 days, in vivo GBM formation was
based therapy against tumor cells that have found a
driven exclusively by GSCs but not non-GSCs. These
sanctuary site within the CNS.
1
Department of Dermatology, University Hospital Essen, Essen, Germany
2
Institute for Molecular Medicine, Goethe-University, Frankfurt, Germany
3
Molecular RNA Biology & Cancer, DKFZ and Department of Pathology, University of Heidelberg, Heidelberg, Germany
NKG2D is a receptor of Natural Killer (NK) cells
which leads to p53 activation. Molecular analysis
and different subsets of T lymphocytes, involved
revealed that the negative effect of Nutlin-3a on
in the detection of stressed abnormal self, as it
ULBP2 expression was absolutely dependent on p53
occurs upon infection or malignant transforma-
activation and was, at least in part, due to the p53-
tion. NKG2D binds to multiple surface ligands,
mediated increase of cellular miR-34 levels. Taken
structurally related to classical MHC class I mol-
together our data demonstrate that members of the
ecules that belong to either the MIC (MICA, MICB)
tumor-suppressive miR-34 family and the tumor
or the ULBP (ULBP1-6) molecule family. A variety
suppressor p53 control ULBP2 expression levels,
of human malignancies show surface expression
which strengthens the role of this specific NKG2D
of MIC and ULBP molecules, sensitizing them for
ligand in innate anti-tumor immunity.
NK cell- and T-cell-mediated cytotoxicity. However,
tumor cells also escape from NKG2D-dependent
immune responses by proteolytic ligand shedding.
Recently, we demonstrated expression of the
NKG2D ligand ULBP2 on human melanoma cells
in vitro and in situ. Furthermore, we measured elevated levels of shed soluble ULBP2 in sera from
melanoma patients that turned out to be an independent predictor of poor prognosis even in early
disease stage. Based on these findings we asked
for the regulation of ULBP2 expression in melanoma cells. We observed that the tumor-suppressive microRNAs (miRNAs) miR-34a and miR-34c
directly control ULBP2 expression. Transfection
of tumor cells with specific miR-34 inhibitors increased ULBP2, whereas transfection of miR-34
mimics downregulated ULBP2 levels which in turn
diminished tumor cell recognition by NK cells.
Treatment of tumor cells with the small molecule
inhibitor Nutlin-3a also decreased ULBP2 expression. Nutlin-3a disrupts the MDM2-p53 interaction
269
215 | Tumor Biology and Interaction with the Immune System
216 | Tumor Biology and Interaction with the Immune System
Regulation of natural killer cell development and antitumor
immunity by the interleukin 15 system
Zoledronate Nanoparticles Repolarize Neutrophils in Tumor
Microenvironment to Impair Growth of Tumors
Gilbert A. Lee, Yae-Huei Liou, Szu-Wen Wang, Si-Tse Jiang, Nan-Shih Liao
Sibel Mete , Sushil Kumar , and Reto Schwendener
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
1
1
1
1
Institute of Molecular Cancer Research, University of Zurich, Winterthurerstr. 190, Building Y
17, Floor K, CH-8057 Zürich, Switzerland
Natural killer (NK) cells are innate immune cells
Zoledronate, a drug used in osteoporosis and bone
nate, as their depletion renders zoledronate ineffec-
involved in tumor surveillance. The development
metastases, is known to restrict tumor growth
tive in restricting tumor growth.
and effector function of NK cells require interleu-
by virtue of its capacity to inhibit angiogenesis.
Furthermore, we found that administration of
kin 15 (IL-15) signal, which is “trans-presented” to
Notably, a large percentage of tumors are resist-
recombinant
IL-15 receptor (IL-15R)βγ on NK cells by IL-15Rα on
ant to angiogenesis inhibitors and therefore require
animals reduced the therapeutic efficacy of the
neighboring cells. Because the IL-15Rα intracellu-
alternative therapeutic approaches. Here we show
drug by impairing the increased neutrophil influx
lar (IC) domain has the capacity to recruit signaling
that zoledronate in nanoparticles (nanozol) inhib-
in the tumors. Accordingly, in cell culture-based
molecules, we generated knock-in (KI) and trans-
its growth of tumors that are refractory to anti-
assays, we showed that TGF-beta levels influence
genic (Tg) mice that lack the IC domain to assess in-
angiogenic drugs by polarizing neutrophils to the
the neutrophil chemotaxis towards tumor derived
dependently the role of the IL-15 trans-presentation
anti-tumorigenic N1 phenotype.
factors.
level. In this study, we found that the IL-15Rα level
Nanozol inhibits tumor growth in two different
Collectively, our findings reveal novel antitumo-
on bone marrow-derived dendritic cells (BMDCs)
syngeneic mouse tumor models namely Lewis lung
rigenic properties of zoledronate and nanozol that
generated by KI and Tg mice determines the level
carcinoma (LLC) which is refractory to anti-angi-
may serve as a basis for the design of more effective
of Stat5 phosphorylation in NK cells, thus offer-
ogenic drugs and MC-38 colon carcinoma. Treat-
immunotherapeutic approaches against chemore-
ing the opportunity to study quantitative effects
ment with nanozol was more effective in inhibiting
sistant tumors.
of IL-15 trans-presentation on NK cell development
tumor growth in both models when compared with
and function in vivo. We also found that NK cell
free zoledronate. Both zoledronate and nanozol
homeostasis, mature NK cell differentiation, and
treated tumors showed an increase in apoptotic cell
acquisition of effector functions require different
death, accounting for the decreased growth rate.
levels of IL-15 trans-presentation input to achieve
Upon extensive analysis, we identified a marked-
full status. In order to investigate the effects of
ly expanded population of neutrophils in tumors
IL-15 trans-presentation level in anti-tumor immu-
treated by zoledronate and its nanoparticles.
nity in vivo, we established the tumor inoculation
Differential expression analyses revealed that these
experiment. Our preliminary data showed that the
neutrophils regained an N1-type antitumoral phe-
-/-
notype, showing an increased expression of pro-
mice in comparison to WT mice. We will assess the
inflammatory and immunostimulatory mediators.
role of the IL-15 system in anti-tumor immunity.
Furthermore, these cells displayed an enhanced
growth of B16/OVA melanoma increased in IL15
TGF-beta
to
zoledronate-treated
+
ability to stimulate CD8 T-cell proliferation and
IFN-γ production. Study of pharmacological inhibition of neutrophils confirmed that neutrophils are
essential for the antitumorigenic effects of zoledro270
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